Staphylococci

Charlie P. Cruz
Course Facilitator

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 Introduction
 Gram-positive cocci (GPC) are common isolates
in the clinical microbiology laboratory.
 Most GPC are members of indigenous
microbiota.
 Some GPC are causative agents of serious
infectious disease—the focus of this USM.

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General Characteristics

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Catalase Reaction and Appearance
 Members of the staphylococci
 Catalase positive
 On stained smears appear singly, in pairs, and in
clusters often referred to as “bunches of grapes”
 Morphology
• Appear creamy, white, or, rarely, light gold
• Buttery looking
• Some species produce β-hemolysis
 S. aureus

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 Other Characteristics
 Characteristics
 Nonmotile
 Nonspore forming
 Non-encapsulated
 Aerobic or facultative anaerobe
• One exception S. saccharolyticus (obligate anaerobe)
 Rare strains
 Small colony variants (SCVs)
• 1/10 size, some are β-hemolytic

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Micrococcus-Resemble Staphylococci
 Micrococcus luteus  Micrococcus growing
 GPC on sheep blood agar
 Catalase positive
• Note: modified catalase
(SBA)
containing a mild
detergent
 Coagulase negative
 Morphology
 Distinct yellow
pigmented colony
 Generally considered a
nonpathogen

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Differentiating Staphylococci and
Micrococci

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 Coagulase
 Staphylocoagulase enzyme clots plasma.
 Coagulase-positive staphylococci
 S. aureus: human pathogen
 S. delphini, S. intermedius, S. lutrae, S. hyicus:
animal pathogens
 Coagulase-negative staphylococci (CoNS)
 S. epidermidis: hospital-acquired infections
 S. saprophyticus: urinary tract infections (UTIs) in
young, sexually active females
• Important in urine samples

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CoNS-Clinical Source and Significance

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Clinically Significant Species

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 Virulence Factors of S. aureus
Enterotoxins
 Heat-stable exotoxins that cause diarrhea and
vomiting
 Toxins A through E and G through J (nine total)
 Resistant to gastric acid and associated with food
poisoning
• A, B, and D are associated with staphylococcal food
poisoning.

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 Virulence Factors of S. aureus
TSST-1 and Exfoliative Toxin
 Toxic shock syndrome toxin-1 (TSST-1)
 Chromosomal-mediated toxin
 TSST-1 and B, C, G, and I are superantigens.
 Causes toxic shock syndrome
 Produced by phage group I
 Exfoliative toxin (epidermolytic toxin)
 Causes the epidermal layer of the skin to slough off
and is known to cause scalded skin syndrome (SSS)
or Ritter’s disease
• Also implicated in bullous impetigo

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 Virulence Factors of S. aureus:
Cytolytic Toxins
 Extracellular proteins that affect red blood cells
(RBCs) and leukocytes
 Hemolysins and leukocidins
 S. aureus produces four hemolysins
 Alpha (α), beta (β), gamma (δ), and delta (γ)
• α-hemolysin lyses RBCs and damages platelets and tissues.
• β-hemolysin shows enhanced activity by acting on the
sphingomyelin of RBC membranes, causing lysis.
 Hot-cold lysin because it works best at 37° C and very well
when stored at 4° C

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 Virulence Factors of S. aureus:
Cytolytic Toxins (Cont.)
 S. aureus produces four hemolysins
 Alpha (α), beta (β), gamma (δ), and delta (γ)
• δ-hemolysin causes injury to cells and leukocytes but is less
lethal.
• γ-hemolysin
 Also called Panton-Valentine leukocidin (PVL)
• Exotoxin kills polymorphonuclear leukocytes.
• Helps prevent phagocytosis
 Often associated with community-acquired infections

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 Virulence Factors of S. aureus:
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

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 Virulence Factors of S. aureus:
Protein A
 Binds the fragment crystallizable (Fc) portion of
antibodies to avoid phagocytosis
 Masking of its immunogenic proteins with host
proteins to look like “self”
 Assists in blocking phagocytosis
 Negates the protective effects of immunoglobulin G
(IgG)

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Virulence Factors of S. aureus

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 Epidemiology of S. aureus
 Primary reservoir
 Nares
 Other reservoirs
 Axillae, vagina, pharynx, and other skin surfaces
 Hospital outbreaks
 Nurseries
 Burn units
 Surgical patients
 Transmission
 Unwashed hands, inanimate objects (fomites)

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 Mode of Transmission and
Predisposing Conditions of S. aureus
 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

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 Infections Caused by S. aureus:
Skin and Wound Infections
 Opportunistic that can be the result of
 Previous skin injuries (e.g., cuts, burns, surgical
incisions)
 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

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 Infections Caused by S. aureus:
Skin and Wound Infections
 Furuncle (boil)

 Carbuncles
 Boils that have multiple lesions and may progress into
deeper tissues

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 Infections Caused by S. aureus:
Skin and Wound Infections (Cont.)
 Folliculitis
 Mild inflammation of a hair follicle or oil gland
 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

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 Infections Caused by S. aureus:
Skin and Wound Infections (Cont.)
 Folliculitis

 Bullous impetigo

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 Infections Caused by S. aureus:
SSS
 Bullous exfoliative dermatitis
 Staphylococcal scalded skin syndrome (SSSS)
 Occurs primarily in newborns and previously healthy
young children
 May occur in adults
• More likely to occur in renal failure patients
• Immunocompromised

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 Infections Caused by S. aureus:
SSS
 Bullous exfoliative dermatitis
 Staphylococcal scalded skin syndrome (SSSS)

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 Infections Caused by S. aureus:
SSS (Cont.)
 Cause unknown
 Symptoms appear due to a hypersensitivity reaction.
 Severity ranges from mild to severe.
 Localized lesion to large generalized area with
profuse peeling of the epidermal layer
 Lasts about 2 to 4 days
 Spontaneous recovery in children
 Adult cases can lead to mortality.

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 Infections Caused by S. aureus:
Toxic Shock Syndrome
 Association with super-absorbent tampons
 Clinical presentation
 High fever
 Rash of trunk spreading to extremities
 Watery diarrhea
 Vomiting
• Dehydration
 Extreme cases lead to hypotension and shock.
 Disseminated intravascular coagulation (DIC)
 Increase in blood urea nitrogen (BUN) and creatinine
 Fatal in 2% to 5% of cases due to multiorgan system
failure

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Infections Caused by S. aureus:
Toxic Shock Syndrome

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 Infections caused by S. aureus:
Toxic Epidermal Necrolysis
 Abbreviated as TEN
 Multiple causes
 Drug induced
 Infections
 Vaccines
• Cause officially not known
 Similar to SSSS
 Treatment with steroids helpful
• Unlike SSSS
 High mortality rate

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 Infections caused by S. aureus:
Food Poisoning
 Toxin, not bacterial growth, causes disease.
 Enterotoxin A, B, D (A and D most common)
 From enterotoxin-producing strains contaminating the
rich foods (mayonnaise)
 Inadequate refrigeration
 Symptoms
 Appear rapidly about 2 to 8 hours after ingesting food
 Usually resolve in 24 to 48 hours, sometimes less
 Nausea, vomiting, abdominal pain, and cramping

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 Infections caused by S. aureus:
Other Infections
 Secondary pneumonia
 After influenza A infection but relatively rare
• High mortality rate
 Bacteremia and endocarditis
 Intravenous (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

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 S. epidermidis
 Predominantly nosocomial infections
 Skin flora gets introduced in catheters, heart valves,
cerebrospinal fluid (CSF) shunts.
 Organisms often produce a slime layer (biofilm) that
helps adherence to prosthetics and avoidance of
phagocytosis.
 Common cause of hospital-acquired UTIs

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 S. 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 (<10,000 CFU/mL), it is still
considered significant.

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 Staphylococcus lugdunensis

 Can cause both community-associated and

hospital-acquired infections
 Can be more virulent than S. aureus and can
clinically mimic S. aureus infections
 Known to carry mecA (that encodes oxacillin
resistance)

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 Staphylococcus lugdunensis (Cont.)

 Important pathogen in infective endocarditis,

septicemia, meningitis, skin and soft tissue
infections, UTIs, and septic shock
 Aggressive in endocarditis, causing valve
replacement
 Infections have high mortality rate.
 Clumping factor can mimic positive coagulase
slide test.

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 Other CoNS
 Opportunistic pathogens
 S. warneri, S. capitis, S. simulans, S. hominis, and S.
schleiferi
• Normal flora but can be involved in endocarditis, septicemia,
and wound infections
 S. haemolyticus
 Common CoNS
 Can be found in wounds, bacteremia, endocarditis
and UTIs
• May have resistance to vancomycin

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Laboratory Diagnosis

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Specimen Collection and Handling
 Requires no special procedures
 Specimens should be taken from the site of infection.
 Cleanse surrounding area to avoid contamination.
 Transport to the laboratory without delay.
 Prevents drying
 Maintains the proper environment
 Minimizes the growth of contaminating organisms

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 Microscopic Examination
 Numerous GPC  Aspirated abscess
 Polymorphonuclear showing GPC and
leukocytes (PMNs) PMNs
 Purulent exudates,
joint fluids, aspirated
secretions
 Aspirate is best.
 If swabs, clinicians
should send two.

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Microscopic Morphology of
Staphylococcus spp.

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 Isolation of Staphylococci
 Grow easily on SBA plates and thioglycolate
 If heavily contaminated, they can be selected
with one or more of the following media:
 Mannitol salt agar (MSA)
 Columbia colistin-nalidixic acid agar (CNA)
 Phenylethyl alcohol agar (PEA)
 CHROMagar Staph aureus
• Selective media

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 Cultural Characteristics
 Round, smooth, white, creamy colonies on SBA
after 18 to 24 hours incubation at 35° C to 37° C
 S. aureus can produce β-hemolytic zones
around the colonies.
 Rarely exhibits yellow pigment with extended
incubation
 Small Colony Variants (SCVs)
 Nonpigmented, nonhemolytic, pinpoint colonies

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S. aureus Growing on SBA

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 Cultural Characteristics
 S. epidermidis colonies are small- to medium-
sized, nonhemolytic, and gray-to-white.
 Some strains may be weakly hemolytic.
 S. saprophyticus produces slightly larger
colonies with about 50% producing yellow
pigment.
 S. haemolyticus grows as medium-sized
colonies with weak or moderate hemolysis and
variable pigment production.

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CoNS on SBA

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 Identification (ID) Methods
 Oxidative–fermentative (O/F) glucose medium
 Most staphylococci ferment glucose
• Micrococci fail to ferment glucose anaerobically.
• O/F testing does not work for all staphylococci.
 Modified oxidase (e.g., Microdase Disk)
 Rapid test to differentiate staphylococci from
microccoci
 Positive for micrococci
 Most staphylococci are negative.

Copyright © 2015 by Saunders, an imprint of Elsevier Inc. 46

Differentiation Among Staphylococci
and other GPC

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 ID Tests: Catalase
 Principle: tests for enzyme catalase
 2 H2O2 in the presence of catalase becomes 2 H2O +
O 2.
 Procedure highlights
 Drop H2O2 onto clean glass slide.
 Using sterile loop or other sterile tool, pick up a
representative colony to be tested.
 Mix and examine for bubbling.
• Bubbling = positive (staphylococci and other bacteria, O2
generated)
• No bubbling = negative (streptococci and other lactic acid
bacteria, no O2 generated)

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 ID Tests: Coagulase
 Clumping factor (formerly cell-bound coagulase)
 Causes agglutination in human, rabbit, or pig plasma
 Slide test method
 Mix water or saline (heavy) suspension of organism with a
small amount of rabbit plasma.
 Check for clumping (if clumping, then positive).

 If clumping is negative, a tube test should be

performed.
 Why?
• Insensitivity of slide test

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 ID Tests: Coagulase (Cont.)
 Test detects extracellular free coagulase.
 Extracellular enzyme secreted that clots plasma
 Tube test
• Check for coagulation 4 hours after inoculation and 24 hours
after, incubate tube at 27° C.
 Prevents autolysis and false-negative result
 Hallmark test for S. aureus
 Other staph can produce positive coagulation.
 Pyrrolidonyl arylamidase (PYR) test is negative for S.
aureus but positive for the other coagulase-positive
staphylococci.

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Tube Coagulase Test

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Coagulase-Positive Staphylococcus:
Clinical Source and Significance

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Key Tests for ID of the Most Clinically
Significant Staphylococci

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 ID Tests: Differentiating CoNS
 If it is a urine sample
 test for S. saprophyticus.
 Presumptive ID can be done using novobiocin (5
μg).
 Streak organism onto plate and add novobiocin disk
to heavy growth quadrant
 If resistant, S. saprophyticus
 If susceptible, likely S. epidermidis
 Can be others, additional testing may be required

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Novobiocin Susceptibility Test

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 Separating Micrococcus
 Taxo A bacitracin disk test
 Micrococcus
• Susceptible (Micrococcus luteus)
• Lemon yellow color of colony does not hurt
 Do not produce acid under anaerobic conditions in glucose O/F
media
 CoNS
• Resistant (S. saprophyticus, S. epidermidis, or others)

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Staphylococcal Schematic

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 Rapid Methods of ID
 BBL™ staphyloslide
 Staphaurex®
 BactiStaph®
 Plasma-coated carrier particles (e.g., latex)
detect both clumping factor and protein A.
 Particularly useful for ID of methicillin-resistant S.
aureus (MRSA), which are weakly positive or
negative in slide coagulase test

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Slide Coagulase Test

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 Rapid Methods of ID
 Real-time polymerase chain reaction (PCR)
 For MRSA and methicillin-sensitive S. aureus (MSSA)
 Qualitative nucleic acid hybridization assays
targeting rRNA sequences for staphylococcal ID
from blood cultures
 Matrix-assisted laser desorption/ionization time-
of-flight (MALDI-TOF) mass spectrometry
 Continues to be integrated as U.S. Food and Drug
Administration (FDA) approval occurs

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 Matrix-assisted laser desorption/ionization time-of-
flight (MALDI-TOF) mass spectrometry

Analysis procedure using MALDI-TOF MS for identification of clinically relevant bacteria. An operator takes
whole cell bacterial material (a) with a toothpick from a pure culture (b) and places bacterial smears on a
steel target (c). The steel target is inserted into the loading port of the mass spectrometer. When the proper
vacuum of 5 x 10 -6 mbar is reached, the target will be moved to the ionization chamber. Using an N 2 -
laser, ions are created by soft desorption which are then accelerated in an electrostatic field (d) and
separated in the flight tube (e). The time of flight (TOF) needed for the ions to reach the detector of the
flight tube (f) is directly related to their mass and forms the basis of subsequent calculations of mass peaks.
Specific identification software (Materials Table) then assigns score values to the mass spectrum fingerprint
of ionized bacterial proteins. 61
Antimicrobial Susceptibility

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 Characteristics
 Standard guidelines issued by Clinical and
Laboratory Standards Institute (CLSI)
 Most S. aureus isolates are resistant to
penicillin.
 β-Lactamase testing should be done.
 Serious infections
 Require susceptibility testing

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 Methicillin-Resistant
Staphylococci
 MRSA
 Methicillin-resistant S. epidermidis
 MRSE
 Community-acquired MRSA (CA-MRSA)
 Can be greater than 50% of isolates in some areas
 Health care–associated/community onset MRSA
(HACO-MRSA)
 Hospital-associated MRSA (HA-MRSA)
 Pose serious threats to health institutions

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 Methicillin-Resistant
Staphylococci (Cont.)
 Infection control
 Barrier protection
 Contact isolation
 Handwashing
 Treat with vancomycin
 Test for susceptibility with cefoxitin disk
 Borderline oxacillin-resistant S. aureus
(BORSA)
 Can separate from MRSA on oxacillin salt agar plate

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 Methicillin-Resistant
Staphylococci (Cont.)
 CLSI document M100
 Recommends cefoxitin to be used to determine
methicillin resistance
 mecA gene
 Encodes penicillin-binding proteins (PBPs)
 Only a small fraction of the population expresses the
phenotype despite having the genetic potential.
 Gold standard
 mecA gene detected by PCR

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 Vancomycin-Resistant Staphylococci

 Vancomycin-resistant S. aureus (VRSA) and

vancomycin-intermediate S. aureus (VISA)
 Isolated in United States (2002)
 Often in patients with underlying conditions
 Important to adhere to infection control practices and
guidelines to hopefully limit the emergence of this
highly resistant organism

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 Macrolide Resistance
 Resistance to the macrolide clindamycin may
not be obvious.
 Erythromycin and clindamycin should have same
resistance patterns.
• If not, perform D-zone test.
 D-zone test
 Use erythromycin and clindamycin disks.
• Growth between disks but not on side of clindamycin disk
 Inducible clindamycin resistance

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D-Test Positive Isolate

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