BACTERIOLOGY LECTURE Other organism that is gram positive but

catalase negative
WEEK 8
 Rothia mucilaginosa, Aerococcus,
Staphylococci
Alloiococcus otits
Characteristics
Test Staphylococci Micrococci
 Family: Staphylococcaceae Modified
 Genus: staphylococcus oxidase - +
 Part of normal flora specially in skin (Microdase)
Brand name
and mucus membrane
Anaerobic


Catalase (+)
Coagulase (+) & some are Coagulase (-)
acid
production
+ -
CoNS Fermenter Oxidizer
from glucose Obligate
 Gram (+) cocci in singly (mag Aero
(fermentation) aerob
kakahiwalay), in pairs and in clusters tolerance:
 Colonial morphology: creamy (pag bago facultative
(except: M.
pa) using blood agar plate anaerob
kristinae
: yellow (buttery looking when the age and M.
of the culture is already more than 24 varians)
hours) using bolood agar plate Anaerobic
Resembles the family: Micrococcaceace
acid
production
+ -
 Genus: micrococcus from glycerol
in
 Catalase (+)
the presence
 Coagulase (-) of
 Gram (+) cocci in pairs, tetrads, and, erythromycin
ultimately, irregular clusters Growth on
 Colonial morphology: yellow using
blood agar plate
Furoxone-
Tween 80–oil
- +
susceptible resistant
 Found in environment and human skin red O Agar
 Colonies: yellow pigment
 Staphylococcaceae and Furoxone- anti
microbial
Micrococcaceace discuss the same time
agent
because they both have same
characteristic
Resistance to R* (-) S (+)
 Microscopic: Both of them are grama bacitracin
positive cocci (0.04 U)
 Biochemical exam: they are both Resistance R (-) S (+)
catalase positive organism lysosome (50-
mg disk)
Resistance to S* (+) R (-)
lysostaphin
(200 ug/mL)
Anti- Susceptibility testing (resistance test)- Staphylococci
determine the effect of antibiotic or anti-
 Staphle meaning “bunches of grapes”
microbial agent against the organism
 They don’t have flagella
Effects: perform during the biochemical exam  nonmotile, non–spore-forming, and
aerobic or facultatively anaerobic
1. Susceptible (sensitive)
EXCEPT S. saccharolyticus (obligate
 Inhibited by the antibiotic
anaerobe)
 Growth: negative
 Normal flora of skin and mucous
 Result: Disk diffusion- meron kang
membranes of human & animals
culture medium, mag lalagay ka ng
 Colonies: medium sized (pinhead) (4 to
organism, then mag lalagay ng anti
8 mm) and appear cream-colored,
biotic disk, nest is incubate at 37°C for
white or rarely light gold, and “buttery-
18-24 hours (LOOKING AT THE
looking”
PRESENCE OF INHIBITION)
 Non fastidious- they can grow with the
2. Resistant
presence of only basic nutrient (CNE)
 Not inhibited
(18-24 HOURS)
 Growth: positive
 Fastidious strains requirements: CO2,
 Result: Walang makikitang zone of
hemin, or menadione with at least 48
inhibition
hrs of incubation

Organisms Infections
S. aureus • cutaneous
infections (folliculitis,
furuncles, carbuncles
& bullous
impetigo)
• food poisoning
• Scalded skin
syndrome (SSS)
• Toxic shock
Staphylococci exemption that is catalase syndrome (TSS)
negative: • Toxic epidermal
necrolysis (TEN)
 S. sciuri
S. epidermidis • nosocomial
 Macrococcus caseolyticus
infections
 S. lentus S. saprophyticus • UTI (in adolescent
 S. vitulus girls & young
women)
Aerotolerance micrococci exemption because
S. haemolyticus • wound
this is fermenters / facultative anaerobe:
• septicemia
 M. kristinae • UTIs
 M. varians • native valve
infections
 S. lugdunensis • catheter-related  α-hemolysin: lyse RBC, damage
bacteremia and platelets and macrophages and causes
endocarditis severe tissue damage incomplete lysin
 β-hemolysin: also known as
Staphylococcus aureus Spingomyelinase C or hot-cold lysin
 complete lysin: acts on sphingomyelin
 Commonly isolated in the plasma membrane of
erythrocytes
Virulence factors:
 : act in CAMP test
1. Enterotoxins  δ-hemolysin: less toxic than α-
 Groups A-E & G-J hemolysin or β-hemolysin
 Staphylococcal food poisoning  γ-hemolysin: (PVL) Panton-Valentine
- Cause by (BAD) leukocidin
 Toxic shock syndrome (TSS)  Staphylococcal leucocidin
- Cause by (BCGI) - (PVL) Panton-Valentine leucocidin
 Staphylococcal pseudomembranous - exotoxin lethal to PMN
enterocolitis (polymorphonuclear cells)
- Cause by (B) - suppresses phagocytosis
 Heat stable exotoxins (100° C for 30 - associated with severe cutaneous
mins) produce outside the cell wall infections and necrotizing
 Interacting with TSST-1: interact with T pneumonia
cells - often associated with community-
2. Toxic Shock Syndrome Toxin-1 acquired staphylococcal infections
 TSS 5. Enzymes
 previously known as enterotoxin F  Coagulase, protease, hyaluronidase &
 superantigen stimulating T-cell lipase
proliferation  Staphylocoagulase- use as aan
 production of a large amount of identification tool
cytokines for the symptoms - S. aureus
 low concentrations: causes leakage by  Hyaluronidase- spreading factor
endothelial cells - S. aureus
 higher concentrations: cytotoxic - hydrolyzes hyaluronic acid present
3. Exfoliative Toxin in the intracellular ground
 Epidermolytic toxin substance that makes up
 Staphylococcal SSS (scalded Skin connective tissues, permitting the
Syndrome) capable of causing ritter spread of bacteria during infection
disease  Coagulase, protease, hyaluronidase &
 Bullous impetigo- cutaneous lesion lipase
4. Cytolytic Toxin  Lipase (normal flora ng skin)
 extracellular proteins that affect red - by both coagulase (+) and CoNS
blood cells and leukocytes - act on lipids present on the surface
 Lysins & leukocidins of the skin
 S. aureus: α, β, γ, δ hemolysins
 6. Protein A Biochemical Tests
 Cellular components in cell wall of S.
Catalase (pag gram positive cocci)
aureus
 bind the Fc portion of immunoglobulin  Principle: catalase mediates the
G (IgG)- antibody breakdown of hydrogen peroxide (30%
- block phagocytosis and inhibit H2O2) into oxygen and water
action of IgG
Result:
Workflow
 (+) bubble formation effervescence- an
Specimen collection (the site of infection is the escape of gas from an aqueous solution
site of specimen collection) - Staphylococci and micrococci
 (-) no or few bubble formation
 From the site of infection with aseptic
technique Biochemical Tests
 No special procedures
Microdase test
 Aspirates: ideal sample
 Swabs: less satisfactory for both culture  Principle: oxidase enzyme reacts with
and smear results the oxidase reagent and cytochrome C
 Cutaneous lesion- aspirate pag wala to form the colored compound,
swab indophenol after 2 mins
- Micrococcus can form idophenol
Direct Microscopic Examination
Result:
 Gram (+) cocci with PMN cells
 (+) development of blue to purple-blue
Culture
color micrococci
 SBA (sheep blood agar), MSA (mannitol  (-) no color change staphylococci
salt agar selective 7.5 NaCl, halophilic),
Biochemical Tests
CAN, PEA, CHROMagar Staph aureus
 18 to 24 hours of incubation at 35° C to Bacitracin test
37° C
 Principle: determine the effect of a
Macroscopic examination small amount of bacitracin (0.04U) on
an organism
 Colonies: hemolytic pattern either beta
or none Result:
 S. aureus: round, smooth, white,
 S: (+) presence of zone of inhibition
creamy colonies on SBA
around the disk
 S. epidermidis: small to medium sized,
 R: (-) no zone of inhibition
nonhemolytic, gray to white colonies

Microscopic examination

 Gram (+) cocci in singly, in pairs and in

clusters
 Majority is in cluster
  inorganic molecule (anaerobic
respiration
 may ph indicator

oxidation fermentation basal medium- merong

carbohydrats + ph indicators (bromthymol blue)

- acid: yellow
- no acid: green
- alkaline: blue
Biochemical Tests
pag tapos mag culture pwede na mag transfer
Oxidation-Fermentation (O/F) reactions sa ofbm tube yung isa lalagyan ng sterile
 Principle: determine whether an mineral oil dahil yun ang mag poprovide ng
organism uses carbohydrate substrates anaerobic environment. Yung isa as is then
incubate 18-24hr at 37°c.
to produce acid by products using OF
glucose medium B- aerobic condition walang mineral oil
Result: glucose, xylose, mannitol, lactose, A- anaerobic merong mineral oil
sucrose and maltose

 (+) yellow medium; acid production

staphylococci
 (-) no change in color; no acid
production micocci
- S. saprophyticus
- S. auricularis
- S. hominis
- S. xylosus
- S. cohnii

*except M. kristane and M. varians

Oxidation-Fermentation (O/F) reactions

 Fermentation
 GLUCOSE glycolysis PYRUVIC ACID;
end product: acid or acid with gas
 single acid (homolactic acid fermenters)
 mixed acids (lactic acid, propionic acid
& succinic acid) Biochemical Tests
Oxidation Coagulase Test
 GLUCOSE glycolysis PYRUVIC ACID  Principle: used to differentiate
CO2 Staphylococcus aureus from CoNS by
 requires oxygen (aerobic respiration) detecting enzyme coagulase
 formation of fibrin
Result:  0.5mL of plasma plus colony from
culture
- (+) fibrin clot S. aureus
- (-) no fibrin clot CoNS

Coagulase Test

2 forms of coagulase enzyme:

Bound coagulase/ clumping factor

 bound to the bacterial cell wall and

reacts directly with fibrinogen in plasma  Extend 4hr in 21-25°C
 alteration of fibrinogen precipitates  Need to check every 30 minutes interval
on the staphylococcal cell because maari mag produce si staph ng
 Use for screening fibrinolysin
 Fibrin clot (clumping of cells) (+)  False negative result
S.aureus, S. lugdunensis and S. schleiferi  Extracellular protein enzyme na mag
 Slide coagulate test because you’re rereact dun sa reacting factor na meron
doing this in the slide din sa plasma
 Plasma could be in human, rabbit or pig  Nag popositive lang ditto si S. aureus
plasma in EDTA ideal is rabbit
 Fibrinogen- magkakaroon ng Pyrrolidonyl Arylamidase (PYR) Test
interaction with the bound coagulates  Principle: differentiate S. aureus from
magkakaroon ng clumping other CoNS by the presence of the
 2-3 minutes enzyme L-
pyrroglutamylaminopeptidase
 L-pyrroglutamylaminopeptidase
hydrolyze L-pyrrolidonyl-β-
naphthylamide/PYR (substrate) = β-
naphthylamine
 β-naphthylamine + N, N-
methylaminocinnamaldehyde/p-
dimethylaminocinnamaldehyde
(reagent)
Free coagulase/ Staphylocoagulase Result:
 extracellular protein enzyme (free  (+) Bright red color (Coagulate
coagulase) + CRF (coagulase-reacting negative staph CoNS) S.
factor) lugdunensis, S. intermedius, S.
 coagulase-CRF complex + fibrinogen schleiferi
 fibrin clot possibility of autolysis due to  (-) No color change or an orange
fibrinolysin color S. aureus
 confirmatory
 tube coagulate test
Vogue-Proskauer (VP) test MRSA (methicillin-resistant Staphylococcus
aureus)
 Principle: determine the ability of
some organisms to produce neutral  Penicillin-resistant (β-lactam) strains of
end products from glucose S. aureus
fermentation  Requires penicillinaseresistant
 Acetly-methylcarbinol or acetoin penicillins, such as nafcillin, oxacillin, or
cefoxitin

types:

- CA-MRSA, HA-MRSA. HACO-MRSA

MRSE

TOC:

VISA (vancomycin-intermediate
Staphylococcus aureus)
Result:
VRSA (vancomycin-resistant Staphylococcus
 (+) Red color S. aureus
aureus)
 (-) Yellow color S. lugdunensis, S.
haemolyticus, S. schleiferi Macrolide Resistance

- Resistance to clindamycin
Novobiocin Susceptibility Test - modified double disk diffusion test
(D-zone test)
 Principle: determine the effect of a 5-
μg novobiocin disk on an organism

Result:

 (+) presence of zone of inhibition

around the disk other CoNS
 (-) no zone of inhibition S. saprophyticus

Diagram