ALCANTARA, S.

Chapter 14: Staphylococci

Chapter Outline:

I. General Characteristics
A. Staphylococcus aureus
B. Staphylococcus epidermidis
C. Staphylococcus saprophyticus
D. Staphylococcus lugdenensis
E. Other Coagulase-Negative Staphylococci

II. Laboratory Diagnosis

A. Specimen Collection and Handling
B. Microscopic Examination
C. Isolation and Identification

III. Antimicrobial Susceptibility

A. Methicillin-Resistant Staphylococci
B. Vancomycin-Resistnt Staphylococci
C. Macrolide Resistance
CLINICALLY SIGNIFICANT SPP.
STAPHYLOCOCCI
1. Staphylococcus aureus
2. Staphylococcus epidermidis
I. GENERAL CHARACTERISTICS 3. Staphylococcus saprophyticus
4. Staphylococcus lugdunensis
These organism are normal inhabitants of skin, mucous membranes 5. Staphylococcus haemolyticus
of human as well as the animals.
Angel Evil Satan Lucifer HAIL!
 Catalase-producing
 Gram-positive cocci VIRULENCE FACTORS
 Arranged in pairs, tetrads or clusters.
 Non-motile (No flagella)  Polysaccharide capsule
 Non-spore formers  appear as slime layer or biofilm
 Facultative anaerobes  allows organism to adhere to inorganic surfaces,
 Catalase positive circumventing the action of antibiotics
 Oxidase negative  inhibits phagocytosis
 Grows in 7.5-10% NaCl
 Medium sized (4-8mm) & raised, creamy colonies "buttery  Peptidoglycan
-looking" on blood agar  activates complement and IL1
 Reduce nitrates to nitrites  chemotactic factor for PMNs
 Bacitracin resistant
 Protein A
 surface protein
 Micrococcus spp. - gram-positive cocci arranged in tetrads  bound to cytoplasmic membrane
or appear as clusters which is refereed to as "bunches of  high affinity for Fc receptor on IgG and complement 
grapes" (E.g. Staphylococcus) bind immune active molecules  decreases the ability of
clearance of the organism from the site of infection
 Example of gram  Teichoic acid
positive cocci arranged  provides rigidity to the cell wall
in clusters  mediate binding to fibronectin

 Facultative anaerobes  Extracellular enzymes & Toxins

- They can survive with or without oxygen (except for S.
saccharolyticus which is obligate anaerobe) Coagulase
 conversion of fibrinogen to fibrin
 Catalase test is very important for the staphylococci group  may coat neutrophils with fibrin formed to protect
because it is the test used to differentiate staphylococci from organisms from phagocytosis
streptococci.
Staphylokinase (FIbrinolysin)
Additional Notes:
 dissolves fibrin clots and may enable infection to spread
once clot is dissolved

STAPHYLOCOCCI
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GENERAL  inhibits phagocytosis

CHARACTERISTICS  Peptidoglycan
 gram-positive  activates complement
 arranged in tetrads or and IL1
clusters  chemotactic factor for
 facultative anaerobes PMNs
 medium sized & raised,  Protein A
creamy colonies on  surface protein
blood agar  bound to cytoplasmic
 catalase positive membrane
 oxidase negative  high affinity for Fc
 non-motile receptor on IgG and
 non spore-forming complement  bind
 reduces nitrates to immune active
nitrites molecules  decreases the
 grows in 7.5-10% NaCl ability of
 Bacitracin resistant clearance of the organism
VIRULENCE FACTORS from the site
 Polysaccharide capsule of infection
 appear as slime layer or  Teichoic acid
biofilm  provides rigidity to the
 allows organism to cell wall
adhere to inorganic  mediate binding to
surfaces, circumventing fibronectin
the action of  Extracellular enzymes &
antibiotics Toxins
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Coagulase
 conversion of Staphylococcus aureus

fibrinogen to fibrin 

Most clinically significant/virulent species of staphylococci
Important cause of nosocomial infection
 may coat neutrophils 

Major human pathogen
Coagulase positive

with fibrin formed to  Normal flora of

 anterior nares
 nasopharynx
protect organisms from 

perianal area
skin
phagocytosis  colonizer of mucosa

Virulence Factors
Lipase
 hydrolyzes lipids  Enterotoxins
 colonization of skin and subcutaneous tissues (boils,  Produced 30%-50% of S.aureus isolates
carbuncles, furuncles)  Heat stable exotoxins (Group A-E; G-J)
 Enterotoxin A,B & D: Food poisoning
Hyaluronidase
 These enterotoxins are stable at 100C for 30
 hydrolysis of hyaluronic acid (connective tissues)
minutes, reheating contaminated food does not
prevent disease
Deoxyribonuclease (DNAse)  Cause symptoms for diarrhea and vomiting
 degradation of DNA  Enterotoxin B & C and sometimes G & I: TSS
 Enterotoxin B: staphylococcal pseudomembranous
Exfoliants enterocolitis
 hydrolyze tissue through cleavage of stratum granulosum
in staphylococcal scalded skin syndrome  Toxic Shock Syndrome Toxin-1
 Known as superantigens and have the ability to interact
Enterotoxins A,B,C,C2,D,E,F with many T cells.
 staphylococcal related food poisoning (A &B)  Superantigen stimulating T-cell proliferation and
 toxic shock syndrome toxin-1 (F) / pyrogenic exotoxin the subsequent production of a large amount of
(C) cytokines that are responsible for the symptoms.
Cytotoxins  Activates an aggressive immune response
 Alpha toxin  Causes cases of menstruating-associated TSS.
 disrupts the smooth muscle in blood vessels  Absorbed through vaginal mucosa leading to systemic
 toxic to RBCs, WBCs, platelets, and effects seen in TSS associated with tampon use.
hepatocytes  Previously referred to as enterotoxin F
 Beta toxin
 works with alpha toxin  Exfoliative Toxin
 heat labile spingomyelinase  Known as epidermolytic toxin
 catalyzes hydrolysis of membrane phospholipids  cell  Cause staphylococcal SSS (Scalded Skin Syndrome) or
lysis also known as "Ritter disease"
 Delta toxin  Implicated in bullous impetigo
 Cytolytic to RBCs
 S.aureus, S, epidermidis, S. haemolyticus  Cytolytic Toxins
 Gamma toxin  Destruction of cells (RBCs and WBCs)
 function in association with Panton Valentine  Alpha (), Beta() and Delta() hemolysins: Lysing of
Leukocidin (PVL) RBCs
 Gamma(): Lysing of WBCs;
Additional Notes:  Gamma toxin is also known as Panton Valentine
Leukocidin (PVL)

 Enzymes
 Coagulase (Staphylocoagulase) - Coversion of
fibrinogen to fibrin. (Fibrin = clots)
 Fibrinolysin - destruction or dissolving of clots
 Protease - cleaves protein
 Hyalurodinase - hydrolyzes Hyaluronic Acid
(connective tissue) which causes spreading of bacteria
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 Lipase - hydrolyzes lipids on skin (fats and oils in  Occurs primarily in newborns and previously healthy
sebaceous glands) young children
 Beta Lactamase - breakdown of beta lactam ring in  Caused by staphylococcal exfoliative or epidermolytic
penicillin molecule (not effective on S. aureus) toxin produced by S. aureus
 Adults - occurs in patient w/ chronic renal failure and
 Protein A compromised immune systems
 Binds in the FC portion of IgG  Mortality rate children - 0% - 7% ; adults 50%
 Inteferes in phagocytosis  Duration of disease lasts 2 - 4 days

Epidemiology C. Toxic Shock Syndrome

 Rare but fatal, multisystem disease/destruction
 Primary reservoir for staphylococci is the human naris, with  Symptoms:
colonization occuring in the axillae, vagina, pharynx, and a. Fever
other skin surfaces. b. Chills Progress to
 Transfer of organism may occur c. Vomiting Hypertension/Sh
 Hospital outbreaks can develop d. Diarrhea ock
 Transmission of S. aureus - Direct contact, with unwashed, e. Muscle aches
contaminated hands; contact with inanimate objects f. rash
(fomites)  Two categories:
a. nonmenstruating TSS
Infection and Associated Diseases b. menstruating TSS
 Todd (1978) It is associated with the use of highly
A. Skin and Wound Infections absorbent tampons
 Infection caused by S. aureus are suppurative  Staphylococcal TSS - results from localized infection
 The abscess is filled with pus and surronded by necrotic and by S. aureus (only toxin TSST-1 is systemic)
damaged leukocytes
 Caused by previous injuries such as cuts, burns and surgical *Read book for detailed information (pg. 318)
wounds
D. Toxic Epidermal Necrolysis (TEN)
 Folliculitis  Clinical manifestation with multiple causes
 Mild inflammation of follicle or oil gland  Commonly drug induced; but some is linked to
 Infected area is raised and red infections and vaccines
 Cause is unknown, symptoms appear to be due to a
 Furuncles hypersensitivity reaction
 Can be an extension of folliculitis  Resolved by administration of steroids (initial stage)
 Infected area is large, red, raised and superficial  Mortality rate is high
abscess

 Carbuncles
 Occur when larger, more invasive lesions
develop from multiple furuncles, which can
progress into deeper tissues
 Patients can present with fever and chills,
indicating spread of bacteria.

 Impetigo
 Bullous: caused by S. aureus, larger surrounded
by a small zone of erythema
 Non-bullous: streptococcal, highly contagious
and easily spread by direct contact, fomites and
autoinoculation

E. Food Poisoning
 S. aureus enterotoxin, A (78%), D(38%), and B(10%)
B.
have been associated with gastrointestinal disturbances.
 Due to ingestion of food contaminated with enterotoxin
Scalded Skin Syndrome (SSS) or "Ritter's disease"
producing strains of S. aureus
 Exfoliative toxin (metabolize and secreted by the
 Symptoms shows 2 to 8 hours after consumption (no
kidneys)
fever, just nausea, vomiting, abdominal pain, and severe
 Bullous exfoliative dermatitis
cramping)

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 susceptible to Novobiocin
F. Other Infections  Normal flora of skin and mucous membranes
 Staphylococcal pneumonia
 Known to occur secondary to influenza virus *See table for more detailed information about S. epidermidis
infection
 Rare, and high mortality rate
Virulence Factors
 Develops contagious
 Lower respiratory tract infection or complication
of bacteremia A. Exopolysaccharide "slime" or biofilm
 Enhance organisms adhesion to implanted medical
 Staphylococcal bacteremia device and provide refractoriness to host defense
 Leads to secondary pneumonia and endocarditis
or bone infection Infection and Associated Diseases
 Observed in intravenous drug users
1. Hospital acquired UTI
 Osteomyelitis  Occurs in indwelling catheters
 Occurs as a manifestation secondary to bactermia
2. Prosthetic valve endocarditis
 Infect bones (Diaphysis of the long bones)
 Complication of cardiac valve replacement surgery
 Symptoms: fever, chills, swelling, and pain
around the affected area. Staphylococcus saprophyticus
 Septic athritis
 Associated with UTis in young women
 Frequently caused by S. aureus in children
 Adheres more effectively to the epithelial cells lining the
(trauma to the extremities or history of
urogenital tract than other CoNS.
rheumatoid arthritis or intravenous drug abuse)
 Rare in mucous membranes or skin surfaces
 May or may not be recovered from aspirated
joint fluid.  Urine cultures - it is in low number (<10,000 colony-forming
units/mL)
 Considered a contaminant
 Associates with:
Staphylococcus epidermidis
 pyelonephritis
 Second clinical significant staphylococcus spp.  cystitis in young women and in older men with
 Presence of slime or biofilm indwelling catheters
 Biofilm production - key component in bacterial
pathogenesis; complex interaction bet. host, Morphology/Characteritics
indwelling device, and bacteria  coagulase negative
 Infections caused are predominantly hospital acquired  large, entire, very glossy, smooth, opaque, butyrous, convexx
 Predisposing factors are catheterization, medical implantation, colonies
and immunosuppressive therapy.  usually white but can be yellow or orange colonies
 Common cause of health care-acquired UTIs and Prosthetic  DNAse negative
valve endocarditis  variable growth in MSA (some strains exhibit fermentation)
 Increased frequency in immunosuppressed patients  resistant to Novobiocin
(Septicemia reported in immunocompromised patients)
Virulence Factors
 Infections are associated with the ff:
 intravascular catheters  Adheres to the epithelial cells lining urogenital tract
 cerebrospinal fluid shunts
Infection and Associated Diseases
 other prosthetic devices
 Infections associated with use of implants (indwelling
catheters & prosthetic devices) caused by isolates shown to  Causes UTI (sexually active young females and older omen
produce a biofilm. with indwelling catheters)
Staphylococcus lugdunensis
 Poly--DL-glutamic acid (PGA) - Bacterial factor involved
in adherence of S. epidermidis ; provides protective advantage  Another CoNS
against host defenses.  Gives positive clumping factor test results (but has negative
tube coagulase reaction)
Morphology/Characteritics  Causes community-associated and hospital-acquired infection
 gram-positive cocci in clusters  Can be more virulent
 small to medium colonies  Can mimic S. aureus infections
 raised creamy gray-white colonies on BA  Contains gene mecA - encodes oxacillin resistance
 slime producing strains it adhere to the agar surface  Associated with the ff
 non-hemolytic on BA  endocarditis
 positive growth on CNA  septicemia
 grows on MSA but lacks fermentation  meningitis
 cogulase negative  skin and soft tissue infections
 DNAse negative  UTIs

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 septic shock a. Blood Agar - enriched for the isolation of

staphylococcus
Morphology/Characteristics b. Colistin-Nalidixic Acid Selective medium for
 hemolytic c. Phenylethyl Alcohol G(+COCCI)
 medium sized d. Mannitol Salt Agar - Usef for staphylococcus
 small colony isolation; it is a selective and differential
 unpigmented or creamy-yellow-orange B. Cultural Characteristics

Staphylococcus haemolyticus  Staphylococci produce round, smooth, white, creamy colonies

 Commonly isolated CoNS on Sheep blood agar (SBA) after 18-24 hours (standard
 Reported in wounds, bacteremia, endocarditis and UTIs incubation period)
 Vancomycin resitance exists in some S. haemolyticus isolates  Staphylococcus aureus - Beta hemolytic, creamy, butter-
looking colonies.
Morphology/Characteristics
*Note: Identification based on colony morphology should not be
 medium-sizeed colonies
done because the result that you will obtained on the colony
 moderate or weak hemolysis and variable pigment production
morphology will only provide presumptive identification
 butyrous and opaque
 beta-hemolytic Which is why it is in need to proceed to biochemical testing in
order to provide definitive result that will be release later on the
Virulence Factors laboratory. is helpful to provide

 Staphylococcus lugdunensis
 Gene mecA - encodes for oxacillin resistance Aerobic Anaerobic
Organism Catalase Microdase
Growth Growth
 Staphylococcus haemolyticus Staphylococcus + (-) + +
 Vancomycin resistance
Micrococcus + + + (-)
Infection and Associated Diseases
Table 1. Differentiation of Staphylococcus and Micrococcus by
 Endocarditis and UTI utilizing Biochemical Testing.
ADDITIONAL NOTES:
MSA Novobiocin Furazolidone Lysostaphin
Organism Coagulase DNAse Bactiracin
Fermentation (5g) Organism
(0.04U) (100g) (200g/mL)
S. aureus + + + Staphylococcus R S S
S. epidermidis (-) (-) (-) S Micrococcus S R R
S.
saprophyticus (-) (-) (-) R
Table 2. Differentiation of Staphylococcus and Micrococcus
based on its Antimicrobial Susceptibility Testing (R= Resistant;
S= Susceptible)

Table 3. Differentiation between the different Staphylococcus

spp.

LABORATORY DIAGNOSIS 1. Catalse Test

 Test the ability of the organism to breakdown
*For Specimen Collection and Handling and Microscopic H2O2 (Hydrogen peroxide) to oxygen and water
Examination refer on pg. 319-320 of the book  Differentiates micrococcal and staphylococcal
species from streptococcal species
A. Isolation and Identification  Reagent used: Hydrogen peroxide

1. Specimen: Positive Copious bubble formation

 Aspirate or swabs Negative No bubble formation
2. Culture Media:
+ Staphylococcus & Micrococcus
- Streptococcus

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 Used to detect the ability of the organism to

hydrolyze DNA
 When DNA is hydroyze, methyl green is released
and combines with highly polymerized DNA at a
pH of 7.5, turning the medium colorless around the
test organism

Hydrolysis of the surrounding medium (clear zone) -

+ Staphylococcus aureus
No Clearing observed - S. epidermidis & S.
- saprolyticus

5. Mannitol Salt Agar

 Test for the ability to ferment mannitol
 Selective and differential for staphylococcus
 Inoculated (red) : Free from
organisms
 Staphylococcus aureus: Yellow
halos surrounding growth (Growth
w/fermentation)
 Staphylococcus epidermidis: Plate
remains pink to red Growth w/o
Fermentation
2. Coagulase Test
 Detects the ability of bacteria to convert fibrinogen  Components: NaCl, D-mannitol and Phenol Red
to fibrin
6. Bacitracin Susceptibility
 2 types of Coagulase Test:
 Test to the susceptibility to 0.04U Bacitracin (or
Bound Taxo A)
 Uses slide coagulase test  0.04U bacitracin/Taxo A disk placed on 5% BA
 Clumping factor streaked with organism
 Positive: Macroscopic Clumping  Susceptible: Micrococcus (zone diameter > than
 Negative: No clumping 10mm)
Free  Resistant: Staphylococcus (No zone inhibition)
 Uses tube coagulase test
 Extracellular coagulase 7. Novobiocin Susceptibility
 Positive: Clot formation within 1-4 hours  Test for the susceptibility to Novobiocin
 Negative: No clot  Susceptible: S. epidermidis (Zone diameter >
3. Mi crodase 16mm)
Test  Resistant: S. saprophyticus (Zone diameter < to
16mm)

 Test for the position of Cytochrome C]

 Modified Oxidase test
 Used to differentiate gram-positive, catalase-
positive cocci (micrococci from staphylococci)
 Detects oxidase
 in the presence of atmospheric
oxygen, the oxidase enzyme
reacts with the oxidase reagent
and cytochrome C to form the
colored compound, indophenol.

+ Dark Blue/ purple (Micrococcus)

- No Color change (Staphylococcus)

4. DNAse Test

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Table 4. Appearance and Characteristics on 5% Sheep Blood Agar

Source: Bailey and Scott's Diagnostic Microbiology 12th ed. (pg. 257)