Staphylococci
General Characteristics
•Gram positive cocci
• Singly, in pairs, and in clusters
• “Bunches of grapes”
•Morphology
• Appear creamy, white, or rarely light gold
• “buttery looking”
• Some species produce β-hemolysis
• S. aureus
• Compare lab characteristics with characteristics found on p. 374
•Characteristics
• Non-motile
• Non-spore forming
• Non-encapsulated
• Aerobic or facultative anaerobe
• One exception S. saccharolyticus (obligate anaerobe)
Coagulase
•Coagulase positive staphylococci
•S. aureus – Human pathogen
•S. delphini, S. intermedius, S. hyicus, S. shleiferi – Animal
pathogens
•Coagulase negative staphylococci
•S. epidermidis – hospital acquired infections
•S. saprophyticus – UTI’s in young sexually
active females
• Important in urine samples
•Others- mostly considered normal flora
• 33 species
Coagulase-Negative Staphylococci
• S. epidermidis
• S. saprophyticus
• S. haemolyticus
• S. lugdunensis
• S. kloosii
• S. saccharolyticus
• S. simulans
• S. capitis
• S. caprae
• S. sciuri
• S. hominis
• S. schlieferi
• S. cohnii
• S. xylosus
See Table 14-2
Micrococcus
• Micrococcus luteus
• Gram positive cocci
• Catalase +
• Note: Modified catalase containing a mild detergent
• Coagulase –
• Morphology
• Distinct yellow pigmented colony
• Generally considered a non-pathogen
Clinically Significant Staphylococci
•Staphyloccus aureus
• Habitat: anterior nares (carriers) (20-30% of humans)
• Primary pathogen of the genus
•Produce superficial to systemic infections
• Skin
• Bacterial sepsis
•Mode of transmission: traumatic introduction
• Needle stick
• Destruction of skin layers (burns, road rash)
• Medical procedures
•Predisposing conditions
• Chronic infections
• Indwelling devices
• Skin injuries
• Immune response defects
Virulence Factors of S. aureus
•Enterotoxins
•Heat stable exotoxins that cause diarrhea and vomiting
•Toxins A-E and G-I (8 total)
• Resistant to gastric acid and associated with food poisoning
• B, C, and sometimes G and I are associated with toxic shock
•TSST-1 (Toxin F)
• Produced by phage group I
• Causes toxic shock syndrome
• Toxic shock syndrome toxin-1
• TSST-1 and B,C, G, and I are superantigens
•Exfoliative toxin (epidermolytic toxin)
•Causes sloughing off of the skin and is known to cause
scalded skin syndrome or Ritter’s disease
• Also associated with bullous impetigo
Virulence Factors of S. aureus
• Cytolytic toxins
• Hemolysins
• Three main types α, β, δ (there is a γ but generally not important)
• α hemolysin – destroys platelets and tissues
• β hemolysin – shows enhanced activity by acting on the sphingomyelin of RBC membranes
causing lysis
• Hot-Cold lysin since it works best at 37 and very well when stored at 4°C
• δ hemolysin – causes injury to cells and leukocytes but less lethal
• Leukocidins (Panton-Valentine leukocidin)
• Kills polymorphonuclear leukocytes
• Helps prevent phagocytosis
• Enzymes
• Coagulase
• Very diagnostic but importance in virulence is not completely understood
• Hyaluronidase
• Hydrolyzes the hyaluronic acid present in connective tissues helping spread of infection
• Lipase
• Breakdown of the fats and oil created by the sebaceous glands on skin surfaces
• Protein A
• Bind the Fc portion of antibodies to avoid phagocytosis
• Masking of its immunogenic proteins with host proteins to look like “self”
• Assists in blocking phagocytosis
Infections of S. aureus
•Skin and wound infections
• Pus formers
• Furuncle (Boil)
• A painful inflammation of the skin and subcutaneous tissue
• Carbuncles
• Boils that have multiple lesions and may progress into deeper tissues
• Folicullitis
• Infection of the hair follicle
• Bullous impetigo
• Large pustules surrounded by small zone of erythema (redness)
• Spread by direct contact and fomites – highly contagious
•Sometimes occur due to blocked follicles, sebaceous glands,
and sweat glands
Scalded Skin Syndrome
•Extensive exfoliative dermatitis
•Staphylococcal (SSSS)
• More likely to occur in renal failure patients
• Immunocompromised
•Severity ranges from mild to severe
•Localized lesion to large generalized area with profuse
peeling of the epidermal layer
•Lasts about 2-4 days
•Spontaneous recovery in children
•Adult cases can lead to mortality
Toxic Shock Syndrome
• Association with super absorbant tampons
• Clinical Presentation
• High Fever
• Rash of trunk spreading to extremities
• Watery diarrhea
• Vomiting
• Dehydration
• Band shift in PMN’s
• Why?
• PMN’s are fighting bacteria
• Leads to hypotension
• DIC- Disseminated intravascular coagulation
• Increase in BUN and Creatinine
• Fatal in 2-5% of cases due to multiorgan system failure
Food Poisoning
•Toxin not bacterial growth
•Enterotoxin A-D (A and D most common)
•From enterotoxin producing strains contaminating the
rich foods (mayonaise)
•Inadequate refrigeration
•“Evil potato salad or mayonaise”
•Symptoms
•Appear rapidly about 2-8 hours after ingesting food
•Usually resolve in 24-48 hours sometimes less
•Nausea, vomiting, abdominal pain, and cramping
Other infections
•Secondary pneumonia
•After influenza A infection but relatively rare
• But it has a high mortality rate
•Bacteremia and Endocarditis
•IV Drug addicts presenting with fever
•Enter through injection site
•Osteomyelitis
•Occurs secondary to bacteremia and results in bacteria
invading the bone
•Fever, chills, swelling, and pain around the infected area
•Arthritis if bacteria in the joint
Staphylococcus epidermidis
• Predominantly nosocomial infections
• Why?
• Skin flora gets introduced in catheters, heart valves, CSF shunts,
• Produce a slime layer (biofilm) that helps adherence to prosthetics and avoidance of
phagocytosis
• UTI’s
• Increased from 25% of cases in heart valve infection to 50% from ‘70 to
‘80
• 70% mortality
Staphylococcus saprophyticus
• UTI’s in young sexually active women
• Due in part to increased adherence to epithelial cells lining the urogenital
tract
• Not present in other skin areas
• Urine cultures
• If present in low amounts it is still considered significant
Other Coagulase Negative
Staphylococci
• S. haemolyticus
• Second most common coag neg staph
• Can be found in wounds, UTI’s, bacteremia, endocarditis
• Recently noted resistance to vancomycin
• Opportunistic pathogens
• S. lugdunensis
• S. schleiferi
Isolation of Staphylococci
• Grow easily on blood agar plates and Thioglycolate
• If heavily contaminated they can be selected with the following
• Mannitol salt agar
• Columbia colistin-nalidixic acid agar (CNA)
• Phenylethylalcohol agar
• Selective media
Identification Tests: Catalase
•Principle: tests for enzyme catalase
2 H 2 O2
2 H 2O + O2
•Drop H2O2 onto smear
•Bubbling = POS (Most bacteria, O2 generated)
•No bubbling = NEG (Streptococci and other lactic acid
bacteria, no O2 generated)
Coagulase
•Cell bound coagulase (clumping factor
• Clots human, rabbit, or pig plasma
•Slide test method
• Mix suspension of organism with a small amount of rabbit plasma
• Check for clumping (if clumping then +)
•If clumping is negative a tube test should be performed
• Why?
• 5% don’t produce cell bound coagulase
•Extracellular free coagulase
• Extracellular enzyme secreted that clots plasma
• Tube test
• Check for coagulation 4hrs after inoculation and 24 hours after
• Prevent autolysis and false negative result
•Hallmark Test for Staphylococcus aureus
•Other staph can produce positive coagulation but usually do
not exhibit the same colony morphology as S. aureus
Separating Micrococcus
• Bacitracin disk test
• Micrococcus
• Susceptible (Micrococcus luteus)
• Lemon yellow color of colony doesn’t hurt
• Do not produce acid under anaerobic conditions in Glucose O/F media
• Coagulase negative staphylococci
• Resistant (S. saprophyticus, S. epidermidis, or others)
Differentiating Coagulase Negative
Staph
•If it’s a urine sample
• S. epidermidis or S. saprophyticus
•Presumptive ID can be done using Novobiocin (5ug)
• Streak organism onto a blood plate and add Novobiocin disk to heavy
growth quadrant
•If resistant
• ie. Zone size below at resistant level
• S. saprophyticus
•If Susceptible
• Likely S. epidermidis but can be others would use tests in table 14-5
p.377 to differentiate
(error in text)
S. saprophyticus
Antimicrobial Susceptibility
•Non beta-lactamase producing staph
•Use penicillin
•However up to 85-90% of S. aureus are resistant
• Thus do beta-lactamase test
•Always perform susceptibility testing
•Especially in serious infections
• Never know when you might need it to save a life
•MRSA
•We already discussed this
•VRSA and VISA
•Isolated in U.S. 2002
• Resistant and intermediate forms
Streptococcus
and
Enterococcus
Streptococci Morphology
Plate media
Liquid media
Note: gram + in chains
Streptococcus and Enterococcus
• Habitat
• Indigenous respiratory tract
microbial flora of animals and
humans
• Certain species are also found in
the gastrointestinal and urogenital
tracts of humans
• Clinical infections
• Upper and lower respiratory tract
infections
• Urinary tract infections
• Wound infections
• Endocarditis
Hemolytic patterns
•β-hemolysis
•RBC’s are completely lysed resulting in a clear area around the
colony
•α-hemolysis
•RBC’s are partially lysed resulting in a greening of the area
around the colony
• α’ hemolysis has intact zone of RBC’s with hemolysis around them (know
of this but don’t worry about it (viridans strep)
•γ-hemolysis (non-hemolytic)
•RBC’s are not lysed so there is no change in agar color
Also table
15-1
Physiologic Characteristics
•Pyogenic streptococci
•Pus producing
•β- hemolytic
•Virdans streptococci
beta
•No C carbohydrate so no lancefield group
•α-hemolytic
•Enterococci
•Normal flora of human intestine
•Lactic acid streptococci (lactococcus)
•Lancefield group N
•nonhemolytic
alpha
Species
Hemolysis
Group
Antigen
Common
Terms
S.pyogenes
ß
A
Group A
streptococci
Pharyngitis; scarlet
fever
pyoderma; rheumatic
fever; AGN
S.agalactiae
ß
B
Group B
streptococci
Neonatal sepsis;
puerperal fever;
pyogenic infections;
pneumonia; meningitis
S. equisimilis
ß
C
Group C
streptococci
Pharyngitis; impetigo;
pyogenic infections
Alpha or no
hemolysis
( rarely ß )
Alpha (α)or
none
(rarely ß)
D
D
Enterococci
Nonenterococci
Urinary tract infections
Wound infections
Bacteremia;
Endocarditis
Urinary tract; pyogenic
infections;
Endocarditis infections
Pneumococcus
Bacteremia;
pneumonia;
meningitis;
Viridans strep
Endocarditis
Dental caries
E. faecalis
E. faecium
E. durans
S. bovis
S. equinus
S.
pneumoniae
Alpha (α)
hemolysis
Viridans and
Nonhemolytic
S. sanguis
S. salivarius
S. mitis or
nonhemolytic
S. milleri
S. mutans
Alpha (α)
hemolysis
or no
hemolysis
Other species
-
Disease
Association(s)
Lancefield Classification
• Rebecca Lancefield developed technique in 1930’s
• Place streptococci into a dilute acid solution for 10 minutes
• Soluble antigen used to immunize rabbits
• Develop antibodies to the antigen
• Classified several different antigens
(C carbohydrate groups)
• A-D, F, G
Streptococcus and Enterococcus: Cell Wall
Structure
•Thick peptidoglycan layer
•Teichoic acid
•C=carbohydrate layer
present except in viridans
group
•Capsule in S. pneumoniae
and in young cultures of
most species
Identification Schema
PYR or
or hippurate
Schema to differentiate Group A and B from
other β-hemolytic streptococci
Biochemical Identification
•Susceptibility tests
•Bacitracin (0.04 units) or
“A” disk
• Identifies Group A
streptococci
Can use PYR disk as a
Better test because reaction is
more definitive
Group A streptococcus is susceptible
to “A” disk (left)
See Procedure 15-1
Biochemical Identification
•Susceptibility test
•Trimethoprim
sulfamethoxazole (SXT)
• Inhibits beta-hemolytic
streptococcal groups other than
A and B
Group A streptococcus growing in
the presence of SXT
Biochemical Identification
•Christie-Atkins,
Munch-Petersen (CAMP)
test
•Detects the production of
enhanced hemolysis that
occurs when β-lysin and the
hemolysins of Group B
streptococci come in
contact
• Also can use purified
beta-lysin on confluent strep
colony
• Disks containing beta-lysis also
work
Group B streptococci showing the
classical “arrow-shaped hemolysis
near the staphylococcus streak
See procedure 15-2
Biochemical Identification
•Hydrolysis
See procedure 15-3
•Hippurate hydrolysis
• Differentiates Group B streptococci from other beta hemolytic
streptococci
• Group B streptococci hydrolyzes sodium hippurate
•Add ninhydrin reagent to get purple complex
Biochemical Identification
•PYR hydrolysis
•Substrate L-pyrrolidonylβ−napthlyamide (PYR) is
hydrolyzed by Group A
Streptococci and
Enterococcus sp.
•As specific as 6.5% NaCl
broth for Enterococcus sp.
•More specific than
Bacitracin for Group A
streptococci
PYR test for Group A
streptococci and enterococci.
Both are positive for this test
(right); left is a negative result
See procedure 15-4
Identification Schema
Schema to differentiate S.
pneumoniae from other
α-hemolytic streptococci
Also separates Enterococcus
and Group D streptococci
Laboratory Diagnosis:
Streptococcus pneumoniae
•Identification
•Optochin-susceptibility-te
st–susceptible
• 14mm with 6mm disk
•Bile-solubility-test–positiv
e
• If zone size is irregular
• Will turn a growing culture
of S. pneumo clear (lyse
bacteria)
Biochemical Identification
•Bile Esculin hydrolysis
•Ability to grow in 40% bile and
hydrolyze Esculin are features
of streptococci that possess
Group D antigen
•Enterococci
•Growth in 6.5% NaCl broth
•Differentiates Group D
Streptococci from Enterococci
•Enterococci grows
Both Group D streptococci and
enterococci produce a positive
(left) bile Esculin hydrolysis
test.
See procedure 15-5
Identification Schema
Salt broth procedure 15-6
Schema to differentiate Enterococcus and Group D
streptococci from other nonhemolytic streptococci
Streptococcus pyogenes (Group A)
•M protien
• Essential for virulence
• Evasion of phagocytosis
• 80 different types
• Ex. M5, M10
• Resistance to infection is related to M protein antibody production
•Streptolysin O
• Responsible for hemolysis on BAP
• Oxygen labile- only active under anaerobic conditions
• Destroys WBC’s, platelets, RBC’s, and other tissues
• Can use anti-streptolysin O tests to check for recent exposure
•Streptolysin S
• Oxygen stable
• Can lyse RBC’s and WBC’s
Streptococcus pyogenes (Group A)
• DNases A-D
• Help destroy foreign DNA by excreting it into surrounding area
• Hyaluronidase (spreading factor)
• Breakdown of connective tissue
• Erythrogenic toxin
• Causes a red spreading rash
• Types A, B, C are known
Streptococcus pyogenes (Group A)
•Clinical Infections
• Pharyngitis
• Strep throat
• Scarlet fever
• Erythrogenic toxin
• Rash appears on the chest and spreads to the trunk and limbs lasting 5-7 days
• After rash skin desquaminates
• Skin infections
• Through bites or abrasions to the skin
• Impetigo
• Usually in very young children
• Erysipelas
• Skin infection with a spreading red rash with a demarcated but irregular edge
• Mostly elderly
• Cellulitis
• Deep invasion of GAS leading to necrosis and gangrene
• Sepsis
Streptococcus pyogenes (Group A)
• Complications
• Rheumatic fever
• Occurs after pharyngitis
• Inflammation of the joints, heart blood vessels, and subcutaneous tissues
• Can cause serious damage to the heart valves
• Acute glomerulonephritis (AGN)
• Can occur after pharyngitis or cutaneous infection
• Immunologic mechanisms lead to antigen antibody complexes resulting in damage to
the kidneys
Toxic Infections
• Streptococcal toxic shock syndrome
• Rare but results from the toxin associated with scarlet fever
Streptococcus agalactiae
(Group B)
•Three major serotypes
•I, II, III
• Refer to capsular antigens (polysaccharides)
•Virulence factors
•Capsule- preventing phagocytosis
•Sialic acid appears to be important factor in inhibiting
alternative pathway of complement
•Hemolysin
•DNases
•CAMP factor
•Hyaluronidase
•Protease
Streptococcus agalactiae
(Group B)
•Clinical infections
•Invasive disease in newborns
• Early onset
• <7 days old
• 80% of cases vertical transmission from mother during birth
• Premature birth, membrane rupture, develops into pneumonia or
meningitis with bacteremia
• Results in very high mortality if not treated quickly
• Late onset
• 7 days old and up
• Usually 1week -3 months
• Primarily meningitis
•Adult infections
• Mother after childbirth or abortion
• Endometritis, or wound infections
• Elderly
• Immunodeficiency
Streptococcus equi, bovis
(Group C(G), D and Enterococcus)
• Animal pathogens that sometimes infect humans
• Group D sometimes manifest infections
• Endocarditis
• UTI’s
• Wound infection or abcesses
• Distinguish group D from Enterococcus
• Enterococcus is resistant to penicillin
• Group D is generally susceptible
• Enterococcus is usually associate with UTI’s
Streptococcus pneumoniae
• Capsular antigens
• Contain 82 known types
• About a dozen are most virulent
• Generally if no capsule then avirulent
• Virulence factors
• Capsule
• Hemolysins
• Immunoglobulin A protease
• Neuraminidase and hyaluronidase
Clinical infections of Streptococcus
pneumoniae
•Common as a normal flora in URT and as a pathogen
•Clinical infections
• Pneumonia
• Prevalent in elderly or with other diseases
• Generally occurs as a secondary infection
• Alcoholism, anesthesia, malnutrition, or after other viral infections
• Aspiration of organism
• Lobar pneumonia
• Chills, cough, dyspnea (shortness of breath)
• Leads to edema of the lungs and drowning in own fluids
• High mortality even with treatment (5-10%)
• Sinusitis
• Otitis media
• These from children under 3 yrs old
• Bacteremia
• Meningitis
• These follow the previous infections creating spread over a wide area of the
body
Other infections
•Can also be involved in:
•Endocarditis
•Peritonitis
• All from dissemination
•Generally treat with penicillin
•But if resistant use erythromycin or chloramphenicol
•Vaccine
•Against most common capsule antigens
•Recommeded for asplenic individuals, elderly, cardiac
patients
•Helps reduce incidence and severity
Streptococcus like Organisms
• Examine Chapter 15 p.406-407
• Generally not major pathogens
• Examine for boards but we will not spend extra time on these
• If you have questions please ask
Aerococcus and Leuconostoc
• Leucine aminopeptidase (LAP)
• Smear organism on damp LAP disk
• Incubate 5 min
• Add PEP reagent (prolyl endopeptidase)
• Hydrolysis of Leucine p Naphthylamide
• releases pure naphthylamide
• red after adding PEP reagent