Name: Lenard S.

Abo
Course: III BMLS  COAGULASE TEST
- definitive test to differentiate Staphylococcus
MIDTERM species
WEEK 1 (STAPHYLOCOCCI) A. POSTIVE TEST RESULT
-is a clot formed in tube containing plasma due to
CLINICAL SIGNIFICANT STAPHYLOCCOCI SPP. Staphylocoagulase.
1. S. Aureus -Coagulase + staphyloccus
2. S. Epidermidis 1. S. Aureus
3. S. Saprophyticus 2. S. Intermedius
4. S. Lugdunensis 3. S. Pseudintermedius
4. S. Hyicus
General Characteristics 5. S. Delphini
 From Greek term staple meaning ‘’bunches of 6. S. Lutrae
grapes’’. 7. S. Agnetis
 From family Staphylococcaceae 8. Some strains of S. Schleiferi
 Catalase positive, gram positive cocci  NOTE:
 In stained smears, spherical cells S. lugdunesis and S. schleiferi B occasionally
(0.5 -1.5um) in singly, pairs and clusters. mistaken for coangulase positive because of the
 Non motile spore forming and aerobic or facultative presence of
anaerobs. CLUMPING FACTOR
 Colonies are produced after 18-24 of incubation and - causes bacterial cells to agglutinate in
are medium sized (4-8 mm) and appeared CREAMED plasma and was the basis of a test known
COLORED, WHITE OR RARELY LIGHT GOLD, AND as SLIDE COAGULASE TEST.
‘’BUTTERY-LOOKING’’. Example of slide coagulase positive:
 Small colony varaiants (SCVs)- S. aureus
- rare strains of staphylococci that are -most clinically significant species
fastidious, requiring carbon dioxide, hemin and -causes various cutaneous and purulent abscesses
menadione for growth. -causes soft tissue infections (Impetigo or cellulitis).
-grow on media containing blood, forming -cutaneous infections progresses to deep
colonies about one tenth the size of wild type strains abscesses (carbuncles).
even after 48hr or more of incubation -common cause of infective endocarditis, toxin
 Some are B hemolyticus induced diseases (food poisoning) and associated
with SSS (scalded skin syndrome) and TSS (toxic
 Staphyloccoci vs micrococci shock syndrome).
Note: Staphyloccoci resembles some members of family
Micrococcaceae such as genus Micrococcus. B. NEGATIVE TEST RESULT
 Micrococci -COAGULASE-NEGATIVE STAPHYLOCOCCI (CoNS)
-are catalase producing coagulase-negative, gram 1. S. Epidermidis (nosocomial infections)
positive cocci found in environment and as microbiota in 2. S. Saprohyticus (UTI predominant in
skin adolescent girls and young women)
-often recovered with staphylococci and can be 3. S. haemolyticus (recovered from wounds,
differentiated easily from COANGULASE-NEGATIVE septicemia, UTIs, native valve infections).
STAPHYLOCOCCI (CoNS). 4. S. Lugdunensis-
-like S. aureus are slide coagulase
 other gram positive cocci recovered from human positive
clinical specimen: -associated with catheter related
Rothia mucilaginosa bacteremia and endocarditis.
Aerococcus -should be identified to spp level to
Alloiococcus otitis ( from human middle ear fluid) derived correct treatment options.

 NOTE: currently 40 species and subspecies are

CoNS found on skin and mucous membranes and
some are on specific sites:
S. capitis (head) and S. auricularis (ear)
CLINICAL SIGNIFICANT SPECIES -exhibited in the CAMP test (CHRISTIE, ATKINS,
AND MUCH PETERSEN to identify B streptococci
3. d-hemolysins
Staphylococcus aureus -found in higher percentage of S. aureus and some
-important cause of nasocomial infections CoNs which are considered less toxic.

Virulence factors B. LEUKOCIDINS

1. Enterotoxins 1. Y-hemolysins
2. Cytolytic factors -found in association with PANTON-VALENTINE
3. Protein A LEUKOCIDIN (PVL)
4. Exfoliative toxins *is an exotoxin lethal to polymorphonuclear
leukocytes. Which supresses phagocytosis and
1. ENTEROTOXINS associated with community-acquired
-heat stable at 100 degrees celcius for 30 min so staphylococcal infections.
reheating contaminated food does not prevent disease.
-causes symptoms including diarrhea and vomiting. C. ENZYMES
-produced by 30% to 50% of S. aureus isolates 1. Coangulase
 Food poisoning (ENTEROTOXIN A, B ,D) -staphylocoagulase considered a virulence marker
 TSS (B, C, G, I) 2. Hyaluronidase
 Staphylococcal pseudomembranous enterocolitis -enzyme hydrolyzes hyaluronic acid present in the
(B) intracellular ground substance ,permitting the spread of
 Toxic Shock Syndrome Toxin-1 (TSST-1) bacteria during infection.
-previously referred as enterotoxin F 3. Lipases
-is a chromosomal mediated toxin that cuases the -produced both by coagulase positive staphylococci
majority of the causes of cases of menstruating and CoNS
associated TSS. -acts on lipids pressent in the surface of the skin
-is a superantigen stimulating T-cell proliferation and particularly fast and oils secreted by sebacious glands.
subsequent production of cytokines responsible for
the symptoms. D. PROTEIN A
-low concentration causes leakage by endothelial -found in the cell wall of S. aureus .
cells and in high concentration are cytotoxic -significant role is its ability to binf the Fc portion of
Note : it is absorbed though vaginal mucosa leading immunoglobulin G (IgG0 which blocks phagocytosis and
to systematic effects seen in TSS associated with negate the protective effects of IgG.
tampon use
2. EXFOLIATIVE TOXINS
-also known as EPIDERMOLYTIC TOXIN EPIDEMIOLOGY
-two types (exfoliative toxin A and B)  Primary reservoir - human nares
-cause RITTER DISEASE  Colonization - vagina, pharynx, axillae, skin surfaces
*known as Staphylococcus SSS causes the Nasal carriage (common in hospital
epidermal layer of skin to slough off patients)
*common in newborns and infants and most  Transmission - direct contact with unwashed,
cases on children younger than 5 years contaminated hands and contact with fomites.
*implicated with bullous impetigo.  Major health concern -methicillin-resistant
3. CYTOLYTIC TOXINS Staphylococcus aureus (MRSA)
-affects Red blood cells and leukocytes by producing  DECOLONIZATION- to reduced colonization for
hemolysins and leukocidins. specific populations such as patients in ICU.

A. HEMOLYSIN INFECTION CAUSED BY Staphylococcus aureus

1. a-hemolysin
-lysed erythrocytes, platelets, macrophage and severe A. SKIN AND WOUND INFECTIONS
tissue damge -abscesses is filled with pus and surrounded by
2. B-hemolysins (Sphingomyelinase C) necrotic tissues and damaged leukocytes.
- acts on sphingomyelin in the plasma membrane or 1. Folliculitis
erythrocytes -mild inflammation of a hair follicle or oil gland, the
-also called as ‘’hot-cold lysin’’. which enhanced infected area is raised and red.
hemolytic activity on incubation at 37 degrees celcius and
subsequent exposure to cold (4 degrees celcius)
2. Furuncles(boils) D. FOOD POISONING
-extensions of folliculitis, are large,raised, superficial -enterotoxins most commonly A, D, AND B have
abscesses been associated with gastrointestinal disturbances.
3. Carbuncles -symptoms appear rapidly (2-8 hrs after ingestion
-occur when larger, more invasive lesions develop of contaminated food) and resolved with in 24 to 48 hrs.
from multiple furuncles -no fever associated with this conditions but
-patients often present with fever chills, indicating nausea, vomiting, abdominal pain, severe cramping are
sytematic spread of bacteria. common.
4. Bullous impetigo
-differ from streptococcus non bullous impetigo in E. OTEHR INFECTIONS
that staphyloccocal pustules are larger and surrounded by 1. Staphylococcus bacteremia- associated among
small zone of erythema. intravenous drug users associated with SCVs found on
Note: Non Bullous impetigo contaminated needles.
-also known as impetigo contagiosa 2. Staphylococcus pnemonia- occur secondary to
-is highly contagious infection that is easily spread influenza virus infection.
3. Staphylococcus osteomyelitis- occurs as
B. SCALDED SKIN SYDROMES manifestation secondary to bacteraemia
-is a bullous exfoliative dermatitis that occurs 4. Septic arthritis- frequently caused by S. aureus in
primarily in the new born and previously healthy young children with trauma to extremities .
children. - occurs in patients with history of
-this syndrome is caused by staphylococcal rheumatoid arthritis, diabetes mellitus, recent join
exfoliative or epidermolytic toxin produced by S. aureus. surgery, skin infections, or intravenous drug users.
-occurs commonly in patients with chronic Renal
Failure and immunocompromised patients. Staphylococcus epidermidis
-RITTER DISEASE - form of SSS which are more -is considered normal skin biota but is
extensive generalized condition affecting 90% of the common caused of hospital acquired infections and
body. often contaminant of improperly collected blood
-duration of disease (2 to 4 days) and complete culture specimen.
healing (after 10 days) o=in which toxin is metabolize and -is common caused of health-acquired UTIs
excreted by the kidney. -most commonly caused prosthetic valve
endocarditis
Note: must be differentiated with TEN (toxic -POLY(y-DL-glutamic acid) bacterial factor
epidermal necrolysis) involved in the adherence of S. epidermidis
-has multiple causes but most commonly is
associated with drug reactions and liked to antimicrobials
and anticonvulsives
Staphylococcus saprophyticus
-associated with UTIs in young women
-unknown caused but symptoms appear due to
-second most common cause, after E. coli of
hypersensitivity reaction uncomplicated CYSTITIS.
-TEN can be resolved by administration of STEROIDS -when present in urine may be found in low
early in the initial stage of presentation whereas numbers and can still be considered significant.
STEROIDS aggravates SSS.

C. TOXIC SHOCK SYNDROME Staphylococcus lugdunensis

- rare but fatal multisystem disease characterized by -cause both community associated and hospital
sudden on set on fever, chills vomiting diarrhea,muscle associated infections.
aches and rash which quickly progresses to hypotension -is known to contain the gene mecA, which
and shock. encodes oxacillin resistant
-associated with women using highly absorbent -endocarditis caused by S. lugdunensis is
tampons. particularly aggressive frequently requiring valve
-two categories (Menstruating and non- replacement.
menstruating associated)
-staphylococcus TSS results localized infection while OTHER CoNS
TSST-1 is sytematic 1. S. Warneri
2. S. Capitis
NOTE: S. aureus does not need to be isolated to confirm 3. S. Simulans
4. S. Hominis
diagnosis of TSS
5. S. Achleiferi
 6. S. Haemolyticus - used r species and strains identification
-commonly isolated CoNS D. COAGULASE TEST
-reported in wounds, bacteremia, endocarditis,UTI 1. CELL-BOUND COAGULASE
-some strains are vancomysin resistant - clamping factor causes agglutination at surface of
bacterial cells in human, rabbit, or pig plasma.
7. S. Pseudintermedius -performed on glass slide using heavy suspension of
-common cause of pyoderma In dogs, skin , ear and organism mixed with saline and a drop of plasma .
postoperative infections in dogs and cats and has -if negative , follow up by tube coagulate test
been linked to human. -insensitive test
-many isolates contains two SSCmec elements (II 2. TUBE COAGULASE TEST
and III) resulting in oxacillin resistant. -detects staphylocoagulase or free coagulase which an
extracellular molecule that causes clot yo form when bacterial
cells are incubated with plasma.
SPECIMEN COLLECTION AND -it also reacts with thermostable, thrombin like molecule
HANDLING called coangulase-reacting factor (CRF) to form coagulase-CRF
complex resembles thrombin and indirectly converts
NOTE: Normal skin biota contamination can be
fibrinogen to fibrin.
further reduced by the physician submitting
SECRETION ASPIRATES (best sample) , TISSUE
E. RAPID METHODS OF IDENTIFICATION
SAMPLES OR BLOOD CULTURE rather than swabs.
1. BBL STAPHYLOSLIDE
2. STAPHAUREX
 ISOLATION AND IDENTIFICATION
3. BACTiStaph
-these kits detects both clumping factor (with
A. SHEEP BLOOD AGAR (SBA)
fibrinogen) and protein A in the cell wall of S. aureus
-media where staphylococci can grow easily
(with IgG)
1. Staphylococci- produce round, smooth, white, creamy
-often have higher specificity and sensitivity
colonies after 18 to 24 hours of incubation at 35° to 37° C.
2. S. aureus - can produce hemolytic zones around the
F. MOLECULAR METHODS
colonies and rare yellow pigment.
1. REAL TIME PCR- identify both MRSA and MSSA
3. SCVs- grow as non pigmented, non hemolytic pinpoint-size
2. NON PCR BASED (T2 BIOSYSTEMS AND QVELLA)
colonies mized with colonies exhibiting the normal phenotype.
- detection from patient blood samples with out the need
4. S. Epidermidis- usually small to medium sized nonhemolytic
of 24-48 hrs growth incubation phase.
gray to white colonies
5. S. Saprohyticus - forms sligthly larger colonies with 50 % of
G. MASS SPECTOPHOTOMETRY APPROACHES
the strains producing yellow pigment .
1. MALDI-TOF METHODS
6. S. Haemolyticus- produces medium sized colonies with
-cannot be used to distinguish genetic differences such
moderate or weak hemolysis and variable pigment .
as MRSA and MSSA strains
7. S. Lugdunensis- often hemolytic and medium sized.

B. HEAVILY CONCENTRATED SPECIMEN

1. MSA (MANITOL SAL SUGAR AGAR)
ANTIMICROBIAL SUSCEPTABILITY
NOTE: B-lactamases (penicillinases ) which breaks down
2. CNA (COLUMBIA NALIXIDIC ACID AGAR)
B-lactan ring of many penicillin most S. aureus isolates are
3. PEA (PHENYLETHYL ALCOHOL AGAR)
resistant to penicillin.
C. CHROMagar Staph aureus
-proprietary selective and differential medium for isolation
A. METHICILLIN-RESISTANT STAPHYLOCOCCI
and identification of S. aureus
1. COMMUNITY-ASSOCIATED METHICILLIN-RSISTANT
-futher calssify S. aureus into MRSA (METHICILLIN
STAPHYLOCOCCUS AURESU (CA-MRSA).
RESISTANT S. aureus) and MSSA (METHICILLIN SENSETIVE S.
2. HOSPITAL-ASSOCIATED METHICILLIN-RESISTANT
aureus
STAPHYLOCOCCUS AUREUS (HA-MRSA)
 IDENTIFICATION METHODS
NOTE: VANCOMYCIN- REMAINS TEH TREATMENT OF CHOICE
FOR MRSA INFECTION
A. OXIDATION-FERMENTATION (O/F) RECATIONS
- traditionally used to differentiate staphylococci and
METHOD OD DETECTION:
Micrococci.
1. CEFOXITIN- CLSI recommeded to detect oxacillin
-staphylococci ferment glucose
(methicillin) resistant which si better inducer of
-Micrococci fail to produce acid under anaerobic
mecA-,mediated resistance.
conditions.
-MRSA isolates should be considered resistant to all B-
B. MICRODASE DISK
lactam antibiotics
-modified oxidase test used to rapidly differentiate
-growth of the resistant is enhanced at neutral pH,
Staphylococci (negative) and Micrococci (Positive)
sodium chloride concentration of 2-4%, cooler incubation
temp (30 to 32 degrees celcius) and prolonged incubation (up
C. MALDI-TOF
to 48 hours)
 -MEDIA CONTAINING CEFOTOXIN- used to screen for
MRSA in clinical samples such as nasal specimens.
-after 24 to 48 hours incubation:
MRSA-colored colony
MSSA-inhibit or produce noncolored colonies
-most oxacillin resistant is due to gene mec A
2. LATEX AGGLUTINATION TEST
-used to detect altered penicillin-binding protein (PBP)
-alternative method method for testing and confirmation
of oxacillin resistant.
3. PCR AMPLIFICATION- GOLD STANDARD FOR MRSA
DETECTIONOF THE mecA gene.

B. VANCOMYCIN-RESISTANT STAPHYLOCOCCI

1. VANCOMYCIN- THE DRUG OF CHOICE FOR SERIOUS

STAPHYLOCOCCAL INFECTIONS.
 VANCOMYCIN-INTERMEDIATE STAPHYLOCOCCUS
AUREUS (VISA) RECOVERED IN JAPAN
 VRSA - REPORTED IN UNITED STATE

C. MACROLIDE RESITACE
1. CLINDAMYCIN - frequently used in Staphylococcal skin
infections with addition of MODIFIED DOUBLE DISK
DIFFUSION (D-ZONE TEST)
*useful when discrepant macrolide test results are
obtained (erythromycin resistant and clyndamycin
susceptible).

NOTE: zone of inhibition is observed on the side of clindamycin

disk which looks like letter ‘’D’’