BACT211: CLINICAL BACTERIOLOGY

TOPIC: STAPHYLOCOCCUS SPP.

2ND SEMESTER | S.Y 2023-2024
LECTURER: Ma’am Christy Gonzales
TOPIC - Gram (+) cocci in pairs, tetrads, and ultimately,
SUBTOPIC irregular clusters
SUB SUBTOPIC - Found in environment and human skin
- Colonies: yellow pigment
• Hindi magka-family and nasa magkaibang order ang
• Medically important bacteria/microorganism – micrococcaceae at staphylococcaceae
microorganisms that are causing infection to humans • Other normal flora that resembles staphylococcus:
• There are four focus in the introduction of MLS in - Kocuria spp.
Diagnostic Bacteriology - Kytococcus spp.
- Characteristics • In terms of characteristics, the following species
- Significance resembles staphylococci:
- Laboratory identification - Rothia mucilaginosa
- Anti-susceptibility testing - Aerococcus spp.
• Workflow in Bacterial Identification: - Alloiococcus otitis
1. Specimen Collection
2. Direct Microscopic Examination TEST STAPHYLOCOCCI MICROCOCCI
3. Culture Modified oxidase
Negative (-) Positive (+)
4. Bacterial Identification (Microdase)
- Macroscopic Negative (-)
- Microscopic Except:
M. kristinae
5. Biochemical Test Anaerobic acid
and M.
6. Anti-Susceptibility Testing production
Positive (+) varians
[fermentation] from
STAPHYLOCOCCUS SPP. [facultative
glucose
anerobe;
• Family Staphylococcaceae capable of
- -ceae : family’s name fermentation]
• Catalase (+) Growth on Furoxone-
- Common biochemical test Tween 80-oil red O Negative (-)
Positive (+)
that is associated with agar [no growth;
[not inhibited
staphylococcus [furoxone is an anti- inhibited by
by furoxone]
biotic; incorporated furoxone]
• Coagulase (+) and some are Coagulase (-)
in an agar]
- Coagulase positive ang mga staphylococcus, but
Anaerobic acid
there are some staphylococci that are coagulase
production from
negative (CoNS) glycerol in the Positive (+) Negative (-)
o Coagulase Negative Staphylococcus presence of
• Gram (+) [purple] cocci in singly, in pairs, and in grape- erythromycin
like clusters Resistance to
Resistance+ Sensitive
• Resembles the family micrococcaceae; genus bacitracin (0.04 U)
Micrococcus spp. Resistance to
- Catalase (+) Lysosome (50-mg Resistance Sensitive
- Coagulase (-) disk)

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 Resistance to
Lysostaphin (200 Sensitive+ Resistance
ug/mL)
• Micrococci – Obligate Aerobe – Oxidation; requires
oxygen to grow and for metabolism; use glucose with
the presence of oxygen
• Staphylococci – Facultative Anaerobe – Fermentation;
gumagamit ng organic compounds
• Disk Diffusion Method
- Gagamitan ng MHA or Blood Agar Plate
- Magc-culture ng organism [first step]
o Culture entirely; cover the entire media
with bacteria
- On top of the culture, maglagay ng antibiotic disk • The following bacteria on the top of the table are gram
[bacitracin, lysosome, lysostaphin] (on top of the (+) cocci.
lawn [culture]) • Not all staphylococcus are Microdase negative. The
- Incubate it for about 18-24 hours following bacteria are staphylococcus that are
- Result [hinahanap natin ‘yung zone of inhibition] microdase positive:
o Measure the zone of inhibition - S. sciuri
o Interpretation (+): susceptible/sensitive - Macrococcus caseolyticus
[more than 10mm] - S. lentus
o Interpretation (-): resistant/resistance - S. vitulus

CHARACTERISTICS
• Staphle meaning “bunches of grapes”
• nonmotile, non–spore-forming, and aerobic or
facultatively anaerobic except for S. saccharolyticus and
S. aureus subsp. Anaerobius
- obligate anaerobe and they may be catalase
negative (-)
• Normal flora of skin and mucous membranes of humans
and animals
• Colonies: medium-sized (4-to-8-millimeter [mm]) and
appear cream-colored, white, or rarely light gold, and
“buttery looking” [butyrous]

- Naga-appear daw ang golen yellow kapag older ang

culture [more than 24 hours]
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 - At the beginning, cream colored, white, grayish o Bullous impetigo – these are like chicken
colony pox lesions, but larger; erythema [redness]:
- But, in some reference, even fresh cultures can pustule
appear golden yellow
• Slime layer: glycocalyx
- Staphylococcus aureus and Staphylococcus STAPHYLOCOCCUS AUREUS
epidermidis
o Polysaccharide; appearing as a slime layer VIRULENCE FACTORS
o Kapag nagdikit dikit, nagfform ng biofilm • Groups A-E and G-J
- Serologically identified
▪ Biofilm [capable of adhering to
• Infections associated with
inorganic surface and may be
Enterotoxins:
resistant or may overcome actions
- Staphylococcal food poisoning
of antibiotic] [A-E] (tinitignan ‘yung on-set of
▪ Not antibiotic resistant, but because manifestation; 2-6 hours after
of the formed biofilm, they can consuming the contaminated
protect themselves from the food [S. aureus]) (viruses: mas
antibiotics mabilis; bacteria: mas mabagal)
• Fastidious strains requirements: CO2, hemin [x-factor], - Toxic Shock Syndrome [B, C, G, I]
or menadione with at least 48 hours of incubation - Staphylococcal
pseudomembranous
enterocolitis [B] [contaminated
milk product]
Enterotoxins
• Heat stable exotoxins (100°C for 30
minutes)
- Para madeactivate, dapat more
than 100°C for more than 30
mins.
- More than boiling ang gagawin
- Boiling or heating the food
doesn’t kill the bacteria
- However, we have a threshold
[maximum amount of
enterotoxin before having
clinical manifestation]. So, it
doesn’t mean na may
enterotoxin ay maf-food poison
na tayo.
• Toxic Shock Syndrome [TSS]
- Cutaneous infection [skin and wound infection] • Previously known as enterotoxin F
o Folliculitis – inflammation or infection in • Also known as pyrogenic exotoxin C
the hair follicle [no systemic manifestation] Toxic Shock - has systemic effect [fever,
o Furuncles – boil; formation of pus [no Syndrome hypotension [decreased plasma
systemic manifestation] Toxin-1 volume; can lead to shock and
o Carbuncles – even larger; more invasive death; more affected: women
using tampons during
type of infection than the furuncle; can
menstruation], desquamation]
progress into deeper layer; systemic
• Epidermolytic toxin A & B
manifestation [fever, chills] Exfoliative
• Staphylococcal SSS
Toxin
- Also known as Ritter disease

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[Serine • Bullous impetigo (neutrophil, eosinophil,
proteases basophil)]
splits the - Suppresses phagocytosis
intracellular - Associated with severe
bridges of the cutaneous infection and
epidermis, necrotizing pneumonia
resulting to - Often associated with
burn-like community-acquired
effect or staphylococcal infections
extensive • S. aureus, S. epidermidis, and S.
sloughing of haemolyticus have delta hemolysin.
epidermis • Coagulase, protease, hyaluronidase,
producing and lipase
“burn-like” - Staphylocoagulase [to identify
effect] S. aureus] [also known as the
• Extracellular proteins that affect red free coagulase]
blood cells and leukocytes - Hyaluronidase [known as the
• Lysins [hemolysin; RBC] and spreading factor]
Leukocidins [WBC] o Hydrolyzes hyaluronic acid
• S. aureus: , , , ō hemolysins present in the intracellular
Enzymes
- -hemolysin: disrupts the ground substance that makes
smooth muscle in blood vessels up connective tissues,
and is toxic to erythrocytes, permitting the spread of
leukocytes, hepatocytes, and bacteria during infection
platelets - Lipase
- -hemolysin: also known as o By both coagulase (+) and
SPHINGOMYELINASE C or “HOT- CoNS
COLD LYSIN” o Act on lipids present on the
o acts on sphingomyelin in the surface of the skin
plasma membrane of RBCs • Found in cell wall of the S. aureus
o act in CAMP test [Christie, • Bind the Fc portion of
Cytolytic Atkinson, Munch,Peterson] immunoglobulin G (IgG) and
Protein A
Toxin [identification of complement
or Streptococcus agalactiae] - Block phagocytosis and inhibit
Cytotoxin o Enhances hemolytic activity action of IgG
when exposed to hot [37 • Activates complement, iL-1,
Peptidoglycan
degree Celsius] and cold [4 chemotactic to PMNs
degree Celsius] temperature Teichoic Acid • Mediate binding to fibronectin
(mas nakakasira ng RBC)
- ō-hemolysin: less toxic than a-
hemolysin or B-hemolysin STAPHYLOCOCCUS AUREUS WORKFLOW
- -hemolysin: functions with
I. SPECIMEN COLLECTION
(PVL) Panton-Valentine
leucocidin [work together with • The site of infection is the site of collection
staphylococcal leucocidin to kill • From the site of infection with aseptic technique
PMNs] • No special procedures
• Staphylococcal leucocidin • Aspirates: ideal sample
- PVL (Panton-Valentine • Swabs: less satisfactory for both culture and smear
Leucocidin) results
- Exotoxin lethal to PMN - Direct sa lesion pero dapat malapit sa peripheri
[Polymorphonuclear cells [metabolically active bacteria]
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 II. DIRECT MICROSCOPIC EXAMINATION IVA. MACROSCOPIC EXAMINATION
• Gram (+) cocci with PMN cells • Colonies:
- S. aureus: round, smooth, white, creamy colonies
on BAP
- S. epidermidis: small to medium-sized,
nonhemolytic, gray-to-white colonies
IVB. MICROSCOPIC EXAMINATION
• Gram (+) cocci in singly, in pairs and in grape-like
clusters
• Pwedeng hindi makakita ng bacteria under the
microscope kahit may infection BIOCHEMICAL TESTS
III. CULTURE
• BAP, CAP, MSA, CAN, PEA, CHROMagar Staph aureus • Principle: catalase mediates the
• 18 to 24 hours of incubation at 35°C to 37°C breakdown of hydrogen peroxide
• Propagate the pathogen for easy observation of its (30% H2O2) into oxygen and water
characteristic - 3% H2O2 gagamitin sa lab
- 3% H2O2 → catalase → O2 + H2O
[appearance: bubble formation;
effervescence]
Catalase
• Result:
- (+) bubble formation
[Staphylococcus spp. and
Micrococcus spp.]
- (-) no or few bubble formation
[Streptococcus spp. &
Enterococcus spp.]
• Principle: oxidase enzyme reacts
with the oxidase reagent and
cytochrome C to form the colored
compound, indophenol after 2
Microdase
minutes of exposure or incubation
Test
• Result:
[Modified
- (+) development of blue to
Oxidase Test]
purple-blue color [Micrococcus
• Phenyl Ethyl Alcohol [PEA] – Inhibitor for Gram spp.]
Negative [Selective for Gram Positive] - (-) no color change
• MSA – 7.5% [Bailey’s – 10%] [sundin ‘yung 7.5%] [S. [Staphylococcus spp.]
saprophyticus is also capable in fermenting mannitol] • Example of a susceptibility test
- Test if the bacteria is capable of
resisting or sensitive to the
antibiotic
• Principle: determine the effect of a
Bacitracin small amount of bacitracin (0.04U)
Test on an organism
• Result:
- (+) presence of zone of
inhibition around the disk
[Staphylococcus spp.]
- (-) no zone of inhibition

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 • Principle: determine whether an the aerotolerance is facultative
organism uses carbohydrate anaerobe; kapag ang organism
substrates to produce acid capable of fermenting with oxygen,
byproducts using OF glucose it is obligate aerobe.
medium • Kapag non-utilizer, the
• Result: glucose, xylose, mannitol, aerotolerance of an organism
lactose, sucrose, and maltose cannot be determined
- (+) Staphylococcus spp. [+/+] [asaccharolytic]
- (-)Micrococcus spp. except M. • O/F Result: +/+; +/-; -/-
kristinae & M. [-/-]
• Fermentation
- GLUCOSE → [glycolysis]
PYRUVIC ACID; end product:
o Single acid (homolactic acid
fermenters)
o Mixed acids (lactic acid,
propionic acid and succinic
acid)
• Oxidation • Example, 4A becomes yellow, 4B
- GLUCOSE → [glycolysis] becomes green.
PYRUVIC ACID → CO2 - 4A: Fermenter
- Requires oxygen (aerobic - 4B: negative [no acid]
respiration) - Result: -/+
- Inorganic molecule (anaerobic • If results are suggesting that the genus is
Oxidation-
respiration) staphylococci, proceed to Coagulase Test
Fermentation
(O/F) • Principle: used to differentiate
Reactions Staphylococci aureus from CoNS by
detecting enzyme coagulase
• Result:
- (+) fibrin clot [staphylococcus]
- (-) no fibrin clot [CoNS]
• 2 Forms of Coagulase Enzyme: Slide
• One way to differentiate Coagulase Test [human, rabbit
staphylococcus and micrococcus (ideal), or pig plasma in EDTA]
• OFBM – oxidation-fermentation - Bound coagulase/clumping
basal medium factor
- Contains carbohydrates Coagulase o Bound to the bacterial cell
[glucose, mannitol, lactose, Test wall and reacts directly with
sucrose, etc.] – may partner na fibrinogen in plasma
pH indicator [Bromyhmol blue] o Alteration of fibrinogen →
- Acid: yellow [fermenter] precipitates on the
- No acid: green staphylococcal cell
- Alkaline: blue o Clot (clumping of cells) [this is
• Place sterile mineral oil on top of the what we’re looking for]
culture [if wala, sterile melted o LAS [S. aureus, S. lugdunensis,
petrolatum ang ilagay or sterile and S. schleiferi]
paraffin oil]
- Will provide anaerobic condition
• Kapag ang organism capable of
fermenting with or without oxygen,
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 - Free Coagulase or • Principle: determine the ability of
Staphylocoagulase some organisms to produce neutral
[confirmatory; do not produce end products from glucose
clumping; 5-15% S. aureus] fermentation
Vogue-
[tube coagulase test; preferred - Acetyl-methylcarbinol or acetoin
Proskauer
reagent: rabbit’s plasma in EDTA • Result:
(VP) Test
(o.5 mL) incubate at 37 degree - (+) Red color [S. aureus]
Celsius for 4 hours; monitor - (-) Yellow color [S. intermedius,
every 30 mins. (monitor the S. lugunensis, S. haemolyticus, S.
formation of clot; may schleiferi]
mabubuong clot dahil sa free • SSS Mo [Staphylococcus spp.,
coagulase na magrreact sa Serratia spp., Streptococcus
coagulase na meron sa plasma; pyogenes, Moraxella spp.]
magrereact sa fibrinogen)] • Principle: determine the ability of an
o Extracellular protein enzyme organism to hydrolyze DNA based
(free coagulase) + CRF DNA on the production of
(coagulase-reacting factor) Hydrolysis deoxyribonuclease
o Coagulase-CRF complex • Medium is pale green because of
[indirectly convert fibrinogen; the DNA-methyl green complex
acts as a thrombin] + • Result:
fibrinogen → fibrin - (+) clear/colorless [S. aureus]
o Fibrin clot - (-) green
• Need i-check every 30 minutes • To differentiate CoNS
because of the possibility na mag- • Principle: determine the effect of a
lyse ang cells due to fibrinolysin
5-g novobiocin disk on an organism
[autolytic enzyme]
• >16 mm
• Kapag negative after 4 hrs., extend
• Result:
the incubation [room temperature:
- (+) presence of zone of
21-25 degrees Celsius] for another
inhibition around the disk
20 hrs to complete the 24 hrs.; If
[susceptible]
negative pa rin, report as negative.
o S. epidermidis
• Principle: differentiate S. aureus o S. capitis
from other CoNS by the presence of Novobiocin
Susceptibility o S. haemolyticus
the enzyme L- o S. hominis subsp. Hominis
pyrroglutamylaminopeptidase Test
o S. lugdunensis
• L-pyrroglutamylaminopeptidase → o S. saccharolyticus
[hydrolyze] L-pyrrolidonyl-- o S. warneri
naphthylamide/PYR (substrate) = - - (-) no zone of inhibition
naphthylamine [resistant]
Pyrrolidonyl
• -naphthylamine + N, N- o S. cohnii
Arylamidase
methylaminocinnamaldehyde or p- o S. kloosii
(PYR) Test
dimethylaminocinnamaldehyde o S. saprophyticus [most
(reagent) common]
• Result: o S. xylosus
- (+) Bright red color
[S. lugdunensis, S. intermedius,
S. schleiferi]
- (-) No color change or an orange
color [S. aureus]

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 VISA [VANCOMYCIN-INTERMEDIATE
STAPHYLOCOCCUS AUREUS]

VRSA [VANCOMYCIN-RESISTANT
STAPHYLOCOCCUS AUREUS]

MACROLIDE RESISTANCE
• Resistance to clindamycin
• Modified double disk diffusion test (D-zone test)
• D-zone test – use erythromycin [15 ug] to induce the
resistance to clindamycin [2 ug]
• 15 mm apart
• Gene: erm gene

MRSA [METHICILLIN-RESISTANT
STAPHYLOCOCCUS AUREUS]
• Methicillin – group of antibiotic
- Example: penicillin
• Penicillin-resistant (-lactam) strains of S. aureus
• Requires penicillin-resistant penicillin, such as nafcillin,
oxacillin, or cefoxitin
• Types:
- CA-MRSA, HA-MRSA, HACO-MRSA
- MRSE
- Treatment of Choice: Vancomycin
• Gene: MecA gene
- Better inducer is cefoxitin

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