Staph &

Strep
YEPRMTMD
Objectives:
 Describe the general
characteristics of the genus
Staphylococcus and
Streptococcus
 Describe the virulence
factors associated with
Staphylococci and
Streptococcus.
 Design an algorithm of key
tests to differentiate among
clinically relevant
Staphylococcus and
Streptococcus
 Micrococci
 catalase-producing
 coagulase-negative
 gram-positive cocci found in the environment and as members of the
indigenous skin microbiota.
Tests Staphylococcus Micrococcus
Catalase (+) (+)
Aerobic environment (+) (+)
Anaerobic environment (+) (-)

Modified oxidase test (-) (+)

Glucose utilization Fermentative oxidative
Bactiracin sensi test Resistant susceptible
Lysostaphin sensi test Susceptible Resistant
 O-F test
 Culture medium : O-F medium
 CHO: glucose
 ph indicator: bromthymol blue
 Alkaline : blue
 Acid: yellow

Open tube Sealed tube

( Vaspar/Vaseline)
Staphylococcus yellow yellow
Micrococcus yellow No color change
 Staphylococcus
 “Staphle”- bunches of grapes
 nonmotile, non–spore-forming, and aerobic or facultatively anaerobic
 Colonies produced after 18 to 24 hours of incubation are medium sized (4
to 8 mm) and appear cream-colored, white or rarely light gold, and
“buttery-looking”
 General Characteristics
 catalase-positive
 gram-positive cocci
 singly, in pairs, and in clusters
 Staphylococcus aureus
 suppurative
 toxin-mediated disease
Virulence Factors
 enterotoxins
 cytolytic toxins
 protein A
 exfoliative toxins
 Virulence factors
1. TOXINS
2. Bacterial proteins
3. Factors against immune defenses of the host/enzymes
 1. Enterotoxins
 heat-stable
 diarrhea and vomiting
 stable at 100° C for 30 minutes
 Enterotoxin (A, B,C,C2,D,E,F)
 food poisoning- A, B, and D
 TSS- B and C and sometimes G and I
 Toxic Shock Syndrome Toxin-1
 superantigen, stimulating T-cell proliferation
 chromosomal-mediated toxin
 menstruating-associated TSS approximately 50% of the non
menstruating cases
 Exfoliative toxin
 SSS/ Ritter Dse: cause the epidermal layer of the skin to slough off
 exfoliative toxin A
 exfoliative toxin B
 Cytolytic Toxins
 proteins that affect red blood cells and leukocytes
 four hemolysins:
 alpha - lysing erythrocytes, can damage platelets
 beta-
-(sphingomyelinase C) acts on sphingomyelin in the plasma membrane of erythrocytes
-enhanced hemolytic activity on incubation at 37° C and subsequent exposure to cold (4° C)

 gamma - found only in association with Panton-Valentine leukocidin (PVL)

 Delta- less toxic to cells
 PVL
 exotoxin lethal to polymorphonuclear leukocytes
 invasiveness of the organism by suppressing phagocytosis
 severe cutaneous infections
 necrotizing pneumonia
 2. Enzymes
 coagulase
 protease
 hyaluronidase/spreading factor
 lipase
 Protein A
 its ability to bind the Fc portion of immunoglobulin G
(IgG).
 Binding IgG in this manner can block phagocytosis
 Coagulase
 Promotes fibrin formation around the bacteria thus protecting the
organism from phagocytosis
 Penicillinase
 Disrupts the beta- lactam portion of the penicillin molecule,
inhibiting the action of penicillin
 Hemolysin

 Alpha,beta, gamma, delta

 Destroy RBC, neutron, macrophages, platelets.
 transpeptidase
 or NOVEL PENICILLIN BINDING PROTEIN
 Inhibited by penicillin
Bacterial proteins that destroys
tissues
 Hyaluronidase
 Spreading factor
 hydrolyzes hyaluronic acid present in the intracellular ground
substance that makes up connective tissues, permitting the
spread of bacteria during infection
 Staphylokinase/fibrinolysin
 Dissolves clot formed in response to coagulase
 Clot dissolution will free the organism
 Lipase

 Hydrolysis of lipids on plasma and skin

 Responsible for skin infections
 Infections Caused by Staphylococcus
aureus
1. localized skin infection
 Folliculitis and sty
 Furuncles or boils
 Carbuncles
2.Pyoderma
3. Deep localized infections
 Osteomyletis
 Septic Arthritis
4. Toxinoses
 TSS
 Gastroenetritis
 SSS/bullous impetigo
 Localized skin infection
 Suppurative
 abscess is filled with pus and surrounded by necrotic tissues and
damaged leukocytes
Folliculitis- relatively mild inflammation of a hair follicle or oil
gland; sweat/ sebaceous glands, hair follicles
Furuncles (boils)- extension of folliculitis, are large, raised,
superficial abscesses
Carbuncles: larger, more invasive lesions develop from
multiple furuncles, which can progress into deeper tissues.
 Pyoderma
 Bullous impetigo
 Superficial, spreading, crusty skin lesions
 Or impetigo contagiosa
 Deep Localized infections
 Osteomyletis
 manifestation secondary to bacteremia
 diaphysis of the long bones and establish an infection
 fever, chills, swelling, and pain around the affected area
 septic arthritis
 trauma to the extremities
 history of rheumatoid arthritis, diabetes mellitus, recent joint surgery,
skin infections, or intravenous drug abuse
 Toxinoses
 TSS
-rare but potentially fatal,
 multisystem disease characterized by a sudden onset of fever, chills,
vomiting, diarrhea, muscle aches, and rash, which can quickly
progress to hypotension and shock
 women using highly absorbent tampons
 high temperature, rash, and signs of dehydration, particularly if the
patient has had watery diarrhea and vomiting for several days.
 Food Poisoning
 S. aureus enterotoxins, most commonly
 A (78%),
 D (38%), and
 B (10%)
 infected food handle
 Disease occurs when food becomes contaminated with
enterotoxin-producing strains of S. aureus by improper handling
and storage, which allows growth of the bacteria and resulting
toxin production
 salads, especially salads containing mayonnaise and eggs;
 meat or meat products;
 poultry;
 egg products;
 bakery products with cream fillings;
 sandwich fillings; and dairy products
 Other infections:
 Staphylococcal bacteremia- IV drug user
 Staphylococcal pneumonia - secondary to influenza virus
infection.
 Staphylococcus epidermidis
 normal skin biota
 common source of hospital-acquired infections
-catheterization, medical implantation, and immunosuppressive therapy
 contaminant in improperly collected blood culture specimens
 health care–acquired UTIs
 Prosthetic valve endocarditis
 intravascular catheters, cerebrospinal fluid shunts, and other
prosthetic devices
 Staphylococcus saprophyticus
 associated with UTIs in young women;
 it is the second most common cause, after E. coli, of
uncomplicated cystitis
 Staphylococcus lugdunensis - both community-associated and
hospitalacquired infections
 mecA gene - encodes oxacillin resistance
 pathogen in infective endocarditis,
septicemia, meningitis, skin and soft tissue
infections, UTIs, and septic shock.
 Endocarditis caused by S. lugdunensis is
particularly aggressive, frequently requiring
valve replacement
 Others Staph spp.
 S. warneri
 S. capitis
 S. simulans
 S. hominis
 S. schleiferi
 Laboratory Diagnosis
 Specimen Collection and Handling
 Isolation and Identification
1. Cultural Characteristics
2. Identification Methods
3. Rapid Methods of Identification
 Antimicrobial Susceptibility
Specimen Collection and Handling
 Proper specimen collection, transport, and processing are
essential elements in the correct diagnosis and interpretation of
any bacterial culture result.
 Clinical materials collected from infected sites should be
transported to the laboratory without delay to prevent drying,
maintain the proper environment, and minimize the growth of
contaminating organisms
 Microscopic Examination
 Gram positive cocci in irregular GRAPELIKE CLUSTERS
 ASPIRATE>swab
 Isolation and Identification
 Blood agar medium: smooth, opaque round low convex and
produce yellow colonies- carotenoid pigments
 selective medium – high SALT (7.5%)
 MSA- heavily contaminated specimen
 PEA- inh. Gram negative bacteria ; pinhead white colonies
 Chapman stone agar,
 vogel johnson,
 Columbia colistin nalidixic acid
 Differential and selective

 MSA
-composition
Interpretation of result:
CHOsource : mannitol
mannitol fermenter: yellow
Salt: 7.5 Nacl zone (SAU)
non mannitol fermenter: no
Ph indicator: phenol red color change
( S.epidermidis, S.
saprophyticus)
 Biochemical test
 Catalase: differentiates STAPH from STREP
 Reagent: 3% H202
 Principle: conversion of h202 to h20 and 02 by the enzyme catalase
 Interpretation :formation of bubbles/ effervescence
 (+): staph
 (-) : strep

Note: test should not be done directly on colonies grown on BAP for red
cells contain catalase
 Coagulase:
 Purpose: most useful single criterion for the detection of Staph aureus
 Reagent: fresh human plasma or rabbit plasma with EDTA or citrate
should be used
 Methods:
 Slide:rapid screening test demonstrates cell bound coagulase or the clumping
factor
 Tube: : confirmatory test to all slide tests that are negative in clinically
significant isolates.
-demonstrates the presence of extracellular coagulase or free coagulase

Positive: SAU
Negative: S. epidermidis and sapro
 DNAse

 Purpose: detects the enzyme that degrades DNA

 Culture medium : DNA medium with methyl green
 Interpretation of result:
 Positive clearing of the medium surrounding the organism
 Negative : no clearing of the medium
 Antimicrobial Susceptibility

 NOVOBIOCIN SENSITIVITY TEST

 Purpose: for the presumptive identification of coagulase negative staph or
CONS
 Sensitive: S.epidermidis
 Resistant: S. saprophyticus
 Polymyxin B sensitivity
 SAU :resistant
 S. epidermidisand Sapro : susceptible