Clin.

Usually considered Bacteria of

Microbiology 1 Significance contaminants of
clinical specimen,
this genus are
of primary
Undan, Farah Jayne A.
rarely implicated clinical
INTRODUCTION as cause of significance.
infections. May S. aureus is a
- Family: Micrococceae (Consist of gram positive cause notorious
cocci, arranged in tetrads (Under the microscope), opportunistic in an pathogen. S.
clusters) immunocompromi saprophyticus
- Genus: Staphylococcus sed host. cause
- Term “staphylococcus derived from Greek: honeymoon
o Staphyle= bunch of grapes (Arrangement) cyctisis
Lysostaphin Not lysed with Lysed with
o Kokkos= berry, meaning bacteria occurring
Sensitivity Test lysostaphin lysostaphin
in the grapelike clusters or berry (resistant) (Sensitive)
- They are common to be isolated in a laboratory Furazolidone Resistant to Susceptible to
- Infection that cause by this genus, such as S. Aureus, Sensitivity test antibiotic antibiotic
epidermidis, saprophyticus, lugdunensis, hemolyticus furazolidone furazolidone
species that are commonly causing human infection. Microdase test Positive Negative
- The name of the different species is based on the Oxygen Grow aerobically Facultative
morphological structure. Requirements (Obligate anaerobe (Can
- anaerobe) use either
aerobic
HISTORY respiration
and/or
- Robert Koch (1878)
fermentation
o First to see staphylococci in pus specimen depending on
- Louis Pasteur (1880) the availability
o First to cultivate on liquid medium of oxygen,
- Sir Alexander Ongston (1881) does not solely
o Named the bacteria as “staphylococcus) depend on
- Von Recklinghausen (1871) aerobic
o First observed in pus respiration for
growth)
- Rosenbach (1884)
Carbohydrate Utilize sugars Fermentative
o Named S. aureus (golden yellow colonies), S.
utilization oxidatively Fermentation.
albus (white colonies) (Glucose, sucrose, They ferment
- Passet (1885) & lactose) or not at carbohydrates.
o S. citreus (lemon yellow colonies) all in the O/F test. Metablic
Utilized sugar waste will
TAXONOMY oxidatively- depend.
- Staphylococcus cocci are gram positive cocci and Through oxidation
classified into 2 families (as per 1986 edn. Of Bacitracin Susceptible to 0.04 Resistant to
Sensitivity Test U of bacitracin 0.04 U of
Bergey’s Manual Systemic Bacteriology):
bacitracin
o Micrococcaceae (GPC, catalase positive) –
- Testing refers to the antibiotic that they are resistant
have 4 genera or susceptible.
 Planococcus -
 Micrococcus
 Stomatococcus STAPHYLOCOCCI
 Staphylococcus
- Resembles some of the some member of the
 Micrococcus and
micrococacceae family. Specifically the genus
staphylococcus can cause
micrococcus
human infection
- Micrococci- catalase producing, coagulase negative,
o Steptococcaceae (GPC, catalase negative)
gram positive cocci that are usually found in the
- Newer edition staphylococci are: environment and as a member of indigenous skin
o Phylum: Firmicutes microbiota.
o Genus I - Often recovered with staphylococci.
o Family V: Staphylococcaceae - Differentiate micrococci and staphylococci through
o Order I: Bacillales coagulase test
o Class III: Bacilli - Stahylococci produce yellow pigment
- Catalase-producing and facultatively anaerobe except
DIFFERENCE BETWEEN STAPHYLOCOCCUS AND
for S. saccharolyticus, which is an obligate
MICROCOCCUS
anaerobe.
Characteristics Micrococcus Staphylococc - Coagulase positive because of the enzyme called
Tests us staphylocoagulase (can be present to other
Morphology Large gram- Gram positive staphylococci species)
positive cocci, cocci, - Non-motile, non-spore forming glucose fermenters
usually arrange in sometimes in - Normal inhabitants of the skin, mucous membrane
tetrads or in pair. pairs and in and intestines
short chains. - Staphylococci associated with human infections are
colonizers of various skin and mucosal surfaces
- Spherical cells that appear in clusters, or sometimes part of all lgG molecules except lgG3. It is
singly not an antigen-antibody specific reaction.
- Culture: BAP-colonies (4mm – 8mm) appear - Virulence factors
creamy, white or light gold, or “Butterly-looking” o Enterotoxins
while other species may have gray colonies; some o Toxic Shock Syndrome Toxin-1
species (S. aureus) may be Beta-hemolytic like S.  Have the special p
aureus. o Exfloitive Toxin
- Current Classification  To some age bracket they are very
o 32 species common, and be able to exhibit
o 15 subspecies disease and infection
o Coagulase positive -> Staphylococcus aureus o Cytolic Toxins
(Considered to be a notorious agent under o Enzymes
staphylococci that create infection to human 
that may be life threatening) o Protein A
o Coagulase negative - Culture Characteristics
 Staph epidermidis o Grow on ordinary media
 Known to cause various o Manifesting diff. characteristic and colors of
acquired or nosocomial the colonies as well
infection o Temp 10-42°C. Optimum is 37°C
 Staph hemolyticus o pH range 7.4 – 7.6
 CAN ALSO BE o Aerobes, some strain of S. aureus are
RECOVERED IN THE facultative aerobes
WOUNDS, can cause o Facultative anerobes
infection in the wound
o Nutrient agar
 Staph saprophyticus
 Mannitol salt agar- Large (2-3mm
 Mainly associated with UTI,
in diameter), circular, smooth, shiny,
isolated in the urine sample
opaque & easily emulsifiable colonies
of adolescent girls/young
 Brain heart infusion agar- Yellow
women
pigment w/c does not diffuse into
 Staph ludgunensis
medium
Commensal flora- benifiting from each  Pigment carotene like lipoprotein
other  Pigment production best at 22°C or
when glycerol monoacetate/milk is
 Staph hominis incorporated into medium.
 Can be found in a area that  Glycerol, monoacetate and milk
can cause infection and is  Blood agar- Beta-Hemolysis is
also rare observed.
 Can cause infection in the  Tryptic soy agar- circular, convex,
sculp and entire margin.
 Staph Auricularis- ear infection o Nutrient slope
 Staph capitis  Oil paint appearance
The species Staphylococcus aureus o Blood sugar
 Marked hemolysis on sheep blood
- Most notorious, responsible for numerous infection. agar
Ranging from mild to life threatening infection.  20-25% co2
- Infection that cause by aureus can be categorized  Rabbit/sheep blood
suppurative or toxin mediated disease. o Mac Conkey Agar
- Toxin, these are substances which are being secreted  Pink colonies due to lactose
by the bacterial cell fermentation
- Can be recovered in any clinical specimen  If there is no pink colors= Non-
- Important cause of nosocomial infection lactose fermenters
- Continous to increase in producing community o Liquid media (If S. aureus)
acquired infection as well as drug resistance.  Uniform turbidity in all area of the
- Morphology broth medium
o Gram-postive, spherical cells, mostly arrange o Selective medium
in irregular grape like clusters.  Salt milk agar
o Polysaccharide capsule is only rarely found  If the S. aureus in diff. media,
on cells. ant organisms can grow in
o The peptidoglycan layer us the major that media
structural components of the cell wall. It is  Salt broth agar
important in the pathogenesis of  Ludlam’s medium (Lithium
staphylococcal infections. Other important chloride, tellurite)
component of cell wall is teichoic acid. - Biochemical Reactions
o Protein A is the major protein component of - Reagent -> Result
the cell wall. It is located on the cell surface o Catalase -> Positive
but is also released into the culture medium o Indole -> Negative
during the cell growth. A unique property of o MR -> Positive
protein A. is its ability to bind to the Fc
 o VP -> Positive
o Urease -> Positive
o Nitrate reduction -> Positive
o Mannitol fermentation -> Postive
o Phosphatase production -> Positive
RELATED INFECTIONS AND DISEASE
- Toxin-induced cases
o Scalded skin syndrome (SSS)- is an
extensive exfoliative dermatitis that occurs
primarily in newborns and previously
healthy children
 Toxin responsible for SSS- Epidermic
o Toxic shock syndrome (TSS)- a rare but
potentially fatal, multi-system disease that
is characterized by a sudden onset of fever,
chills, vomiting, diarrhea, muscle aches, and
rashes, and which rapidly progresses to
hypertension and shock.
o Sometimes cause death
o Example:
 Preformed, heat-resistant enterotoxin
mediates staphylococcal food
poisoning (symptoms in 2-6 hours):
usually self-limiting)
 Exfoliative toxins A and B results in
staphylococcal scalded skin
syndrome; usually in infants and
neonates
 Can cause cutaneous erythema the
profiuos peeling
 Occur in hands and feet then spread
to other parts
 10 days of medication with
antibiotics
- Bacteremia and sepsis
- Urinary tract infection
- Acutes bacterial endocarditis
o Inflammation of the outer pusod
o Enlargement of the heart
- Cutaneous infections
o Previous damage or injury
o Folliculitis- mild inflammation of the hair
follicle or subcutaneous gland
 A small red bump or pimple develops
at the infection sites of the hair
follicle.
 Sty: a sty is a folliculitis affecting
one or more hair follicles on the edge
of the upper and lower eyelid

o Furuncles- are large raised superficial
abscesses which can be an extension of
folliculitis. Commonly known as boils.
 Deep seated infection, originating
from folliculitis, (if infection extends
from follicle to neighbor tissue)
 Causes redness, swelling and severe
pain
 Common found on the neck, armpit
and groin regions.
 Superficial abcesses
o Carbuncles- develop from multiple
furuncles which my advance into the
deeper tissues and cause fever and chills
leading to systemic infections.
 Is an aggregation of infected
furuncles. May form large abscesses.
 It is a large area of redness, swelling
and pain, punctuated by several sites
of drainage pus.
o Impetigo- is a superficial cutaneous
infection that is commonly seen in children
and characterized by crusty lesions and
vesicles surrounded by a red border.
 A very superficial skin infection
common in children usually produces
blisters or sores on the face, neck,
hands and diaper area.
 It is characterized by watery blisters,
which becomes putules and then
honey coloured crust
o Purulent abscess
- Osteomyelitis (Deep Infection)
o Inflammation of bone
o Bacteria can get to the bone via bloodstream
and following an injury
o Clinical features: pain, swelling, deformity,
defective healing, in some case pus flow
o Diagnosis: X-ray, MRI, bone aspirates
- Septic arthritis (children)
- Food poisoning
ENZYME AND TOXINS PRODUCED BY STAPH.
AUREUS
1. Coagulase
o It coagulates the fibrinogen in the plasma.
Promotes the formation of a fibrin layer
around the staphylococci abscess thereby
protecting the bacteria from phagocytosis.
o There are two types:
 Cell-bound coagulase or clumping
factor – is bound to the cell wall and
clots human, rabbit, or pig plasma by
directly converting fibrinogen into
fibrin.
 Unbound or free coagulase – is an
extracellular enzyme that is not
bound to the cell wall and causes clot
formation when bacterial cells are
incubated with plasma.
2. Hyaluronidase (Spreading-factor enzyme)
o it enhances invasion and survival in the
tissue. Breaks down the hyaluronic acid that
is present in the intracellular ground
substances of connective tissues, resulting in
the spread of bacteria.
3. Staphylokinase (Fibrinolysin)
o Causes fibrinolytic activities by dissolving
fibrin clots.
4. Lipase (Fat-splitting enzyme)
o Produced by both coagulase-positive and
coagulase-negative staphylococci. Essential
for bacterial survival in sebaceous areas of the
body and important in the formation of
furuncles, carbuncles and boils.
5. Deoxyribonulease (Dnase) and phosphatase
o Lowers the viscosity of exudates giving the
pathogen more mobility and destroys DNA.
6. β- lactamase
- Breaks down penicillin and other β-lactam drugs.
More than 90% of clinical staphylococci isolates
are penicillin-resistant as a result of enzyme
production.
7. Emterotoxin (Heat-stable)
o Acts as neurotoxins that stimulate vomiting
through the vagus nerve, produced by the
majority of Staph.aureus isolates. Stable to
heating at 100 degree centigrade for 30
minutes.
o Resistant to hydrolysis by gastric and jejunal
enzymes.
o Examples of enterotoxins A,B,C1,C2,D,E and
G to J
o A,B and D are responsible for food poisoning.
8. Leukocidin/Panton- Valentine Leukocidin
(Cytolytic toxin)
o It attacks and kills white blood cells, pore
forming exotoxin that suppresses
phagocytosis and responsible for necrotizing
skin and soft tissue infections.
9. Hemolysin
o Causes anemia and make iron available for
microbial growth.
o There are four (4) types of hemolysin:
 Alpha-hemolysin (α) – predominany
lysine that is produced by
S.aureus.Destroys red blood cells,
platelets, and macrophages, and
causes severe tissue damage.
 Beta- hemolysin (β) – destroys
spingomycelin and RBC around
nerves.It has enhanced haemolytic
activity on incubation at 35 degree
centigrade.
 Gamma- hemolysin ( γ) – less toxic
than α and β –lysins, produced by all
S.aureus strains that cause RBC
injury in culture and produces
edematous lesions.
 Delta-hemolysin (δ) – destroys RBC
and is associated with the Panton-
Valentine leukocidin.
10. Exfoliatin serotypes A and B ( Superantigens)/
Epidermolytic toxins A and B
o Is a serin protease that divides the
intracellular bridges of the epidermidis and
causes extensive sloughing of the epidermis
to produce a burn-like effect on the patient.
o Destroys the stratum granulosum and causes
scalded-skin syndrome (SSS) or Ritter’s
disease.
11. Toxic shock syndrome toxin 1 (TSST-1)/
Enterotoxin F/Pyogenic exotoxin
o A chromosomal-mediated toxin and causes
almost all cases of menstruation-associated
TSS
o Stimulates the production of a large amount
of cytokines that are responsible for the
symptoms.
12. Protein A
o Immunologically active substance that is
found in the cell wall.
DIFFERENTIAL TEST FOR STAPH. AUREUS
1. Coagulase Tests
o Best single criterion of pathogenicity of
Staph. aureus - Reagent: Rabbit plasma
o Anticoagulant: Ethylene diaminetetra-acetic
acid (EDTA)
o Two methods that can be used:
 Slide method- used to screen catalase
positive colonies, detects cell bound
coagulase or clumping factor.
 (+) result : clot or coagulum
formation within 30 seconds.
 Other slide coagulase Staph.
are Staph. lugdunensis and
Staph. schleiferi
 Tube method- considered sensitive
but definitive method, detects
extracellular or free coagulase
 (+) result : clot or coagulum
formation after one to four
hours of incubation.
 Other tube coagulase Staph.
are:
o Staph.hyicus,
o Staph.intermedius,
S.delphini
o S. schleiferi
subsp.coagulans
2. Mannitol Fermentation Test
o Used to differentiate the pathogenic staph.
From non-pathogenic ones.
o Culture medium is mannitol salt agar and pH
indicator is phenol red.
o (+) result: Yellow-colored S. aureus colonies
3. Tellurite glycine agar
o This results in jet black colonies of S.aureus
4. Polymyxin sensitivity tests
o S.aureus is resistant to this test
5. Voges-Proskauer (VP) test
o Differentiates S. aureus from S. intermedius
o (+) result: Pink color
6. Deoxyribonuclease (Dnase) test
o Used to identify pathogenic species of
staphylococci that produces Dnase.
o Culture medium: DNA- methyl green agar
o (+) result: Clear/ colorless zone around the
test organism
METHICILLIN-RESISTANT STAPHYLOCOCCUS
AUREUS (MRSA)
- Resistant to antibiotics such as methicillin, nafcillin,
and oxacillin.
- Can be acquired after a prolonged stay in the hospital,
close contact with individuals who are carriers of the
organism, after-effects of a broad spectrum of
antibiotics treatments, and exposure to nasal
secretions.
- Can be controlled by proper isolation of the organism,
rapid identification of the bacteria, hand hygiene,
treatment of sources, and most importantly, a strict
compliance to infection control programs.
- Three (3) types of MRSA:
1. Hospital-acquired (HA) MRSA
2. Community-acquired (CA) MRSA
3. Health care associated community-Onset
(HACO) MRSA
o (+) Chromogenic test: change in color of
MRSA colonies within 24-48 hours using
CHROM agar against colorless colonies of
non- MRSA
COAGULASE NEGATIVE STAPHYLOCOCCUS
(CoNS)
- S. epidermidis & S. saprophyticus
 o They are coagulase-negative staphylococci o CHROM agar is a selective and differential
o Do not produce exotoxins medium for the isolation of MRSA
o Thus, they do not cause food poisoning or o Staphylococci grow easily in routine culture
toxic shot syndrome. media
- S. epidermidis o CoNS recovered from sterile sites and from
o Part of the normal flora if the skin and sites associated with indwelling devices
mucous membrane. should be considered potential pathogens.
o Cause of hospital-acquired infections o A low colony count for S. saprophyticus urine
o It is involved in indwelling catheters, culture is considered significant.
prosthetic materials, shunts, and surgery. 3. Catalase test – a heme enzyme that catalyzes the
o It is common of prosthetic heart valve decomposition of H2O2 to water and oxygen.
endocarditis o Differentiates staphylococci (+) from
o It also cause nosocomial bacteremia; sepsis in streptococci (-)
neonates peritonitis in patients with renal o Reagent is 3% H2O2 ; (+) presence of bubble
failure; cerebrospinal fluid shunt infections formation or effervescence.
o Often multiple antibiotic resistance- - 4. Coagulase test
methicilin o Reagent: Rabbit plasma ; (+) Clot or
- S. saprophyticus coagulum formation
o Commonly isolated from animals and their 5. Mannitol fermentation test
carcasses. o +) Yellow halo around colonies
o Coagulase and phosphatase – ve, urease & 6. Pyrrolidonyl arylamide (PYR) test
lipase +ve o Differentiates coagulase-positive
o S. saprophyticus is resistant to the antibiotic staphylococci by slide method. (+) Cherry
novobiocin, a characteristics used in lab to red; (-) No color change
distinguish it from S. epidermidis 7. Voges-Proskauer (VP) test
o Infections are almost always community o Differentiate staphylococci by tube method.
acquired. (+) Deep pink
o It causes mainly UTI, particularly in sexually 8. B-lactamase test
active young women. a. Cephalosporinase test – most useful and
o It is the second most common cause of UTI, widely used B-lactamase test
after E. coli in young women accounting for b. Acidimetric method
10-2-% c. Iodometric method
o Quinolones are commonly used in treatment 9. Antimicrobial testing
of S. saprophyticus UTI. o Drugs: Methicillin, oxacillin, cloxacillin and
- Staphylococcus lugdunensis dicloxacillin
o It is CoNS by tube method o Culture media: Vancomycin agar screen plate,
o Can be confused with S.aureus if slide Oxacillin-screen plate,Cefoxitin disk
diffusion, double-disk diffusion test
coagulase method is performed
10. Latex agglutination test
o More aggressive than the other CoNS in
o Used in detecting the clumping factor and
terms of infectivity
protein A.
o Contains the mecA gene that codes for
11. Molecular test
oxacillin resistance
o Used in identification of the mecA gene and
o Related infections include infective
gold standard for MRSA detection.
endocarditis, meningitis, septicaemia, UTI,
and skin and soft tissue infections. Streptococcus
o Novobiocin susceptible CoNS species are : S.
epidermidis, S.capitis, S.haemolyticus, - Species play an important role in the production of
S.hominis, S.lugdunensis, S.saccharolyticus diseases and infection
and S.warneri - There are 17 species in this genera that are catalase
o Novobiocin resistant CoNS species are: negative, gram positive cocci
- Is a non-catalase producers organism
S.saprophyticus, S.cohnii, S.kloosii and
- Streptococcus species
S.xylosus
o Streptococcus
LABORATORY DIAGNOSIS FOR STAPHYLOCOCCI o Aerococcus
o Lactococcus
- Specimens: Aspirated secretions (best sample),
purulent exudates, and joint fluids o Leukonostocs
1. Gram stain o Pediococcus
2. Culture: Media that can be used are the ff: BAP, - Over 100 species
MSA, PEA, CAN, BHI, thioglycollate, and - General characteristics: streptococcaceae
CHROM agar - Catalase negative, gram postitive cocci that is usally
o Colistin-nalidixic agar (CNA) used for arrange in pairs or chains
purulent exudates - Negative
o Phenylethyl alcohol (PEA) agar is enriched
with 5% sheep blood and is selective for
Gram-positive bacteria
o Mannitol salt agar (MSA) and PEA for
heavily contaminated specimens