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2. STAPHYOCCI AND MICROCOCCI
Created @November 12, 2022 11:34 AM
Last Edited Time @January 17, 2023 12:28 PM
Type SEMINAR LECTURE AND NOTES
Created By @louiden
Participants
Identification test for Staphylococci and Micrococci
Biochemical Tests
Catalase Test
To differentiate staph and micro from streptococci
Reagent: 3% Hydrogen Peroxide, Positive Results: Vigorous bubbling.
Use of colonies from BAP will cause FALSE POSITIVE
if you used MSA media you can add directly hydrogen peroxide
Modified Oxidase / Microdase
For identification of Micrococci
Reagent 6% Oxidase
CHEMICAL NAME: TETRAMETHYL-P-PHENYLENEDIAMINE DICHLORIDE IN
DIMETHYLSULFOXIDE
POSITIVE TEST: BLUE
Other Method
2. STAPHYOCCI AND MICROCOCCI 1
Cytochrome oxidase
Reagent: 1% Oxidase
Positive: Purple
Coagulase test
To differentiate S. aureus from other streptococci
1st to be done Slide method: Bond coagulase (CRP) Tube: Free coagulase
Reagent:
Rabbit’s plasma collected in EDTA:
Uses of Citrated Plasma:
WILL CAUSE FALSE POSITIVE
Positive Results: CLOT FORMATION
Tube method will require method at 37 Deg for 4 hours, if (-) incubate further at RT for 16 hours.
Preferred: MSA, CNA, SBA, PEA.
💡 DEFINITE TEST FOR S. AUERUES IS THE COAGULASE TEST.
MSA
REQUIRED MEDIA FOR S. AUREUS
Indicator: Phenol red pH Indicator
DNASE TEST
DETECTS:
SERRATIA & MORAXELLA
HCL preparation method → DNASE AGAR → Positive Results: Clearing of agar around the colonies
Dye Method → DNASE AGAR
Dyes: Methyl Green: CLEAR ZONE
Toluidine Blue: PINK ZONE
BETA-LACTAMASE
Purpose: To detect the ability of the organism to produce beta-lactamase
Use: CEFINASE DISK with NITROCIN SUBSTRATE
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Positive Results
Pink to Red color
💡 S. aureus can produce beta-lactamase/penicillinase
Biochem
Cat +
Coag+
Mannitol fermentation: +
DNASE: +
Beta-lactamase+
If all the tests are negative suspected organisms:
CoNS = S. epidermidis and S. saphrophyitucs
NOVOBIOCIN DISK TEST
differential test for
5 ug
S. saprophyticus: Resistant; S. epidermidis: Susceptible
Staphylococcus - facultative
Micrococcus - Strict aerobes
anaerobe
Aerobic growth Growth +
Anaerobic growth Growth -
Lysosthaphin susceptibility SUSCEPTIBLE RESISTANT
Modified Oxidase test +
Bacitracin susceptibility
furazolidone/FUROXONE SUSCEPTIBILITY
Not fermenters but
Glucose Utilization/OF medium Fermenters
OXIDIZERS
HEMOLYSIS ON BAP BETA HEMOLYTIC GAMMA HEMOLYTIC
IF AN ORGANISM IS AN FERMENTER CAN PRODUCE ACID WITH OR WITHOUT AIR.
IF AN ORGANISM IS AN OXIDIZERS CAN PRODUCE ACID ONLY IN THE PRESENCE OF AIR
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Identification test for Streptococci
LAP TEST (leucine amino peptidase)
Test for catalase
Substrate
LEUCINE-BETA-NAPTHYLAMIDE
Reagent
CINNAMALDEHYDE
Positive results: RED COLOR Negative Results: No color or Slight Yellow
Interpretation: Positive + Could be streptococci
Negative
FOR GROUP A BETA HEMOLYTIC
BACITRATIN DISK TEST/ TAXO A
INITIAL DETECTION OF S. PYOGENES
PYR TEST: detects enzyme Pyroglutamylaminopeptidase or pyrrolidonylamylamidase
USES:
Positive Results:
Group B: Beta hemolytic: S. Agalactiae
CAMP - Christine Atkins Much Peterson
Uses: BAP AS MEDIA, you need a known organism: S. AUREUS
Positive Result: Enhance hemolysis as shown ARROW HEAD ZONE OF BETA HEMOLYSIS es
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💡 Board Exam QUESTIONS!
What is the negative result in the CAMP test: PRESENCE OF HEMOLYSIS BUT NO LONGER
ENHANCE (PRESENCE OF ARROWHEAD)
What is the positive result of CAMP: PRESENCE OF HEMOLYSIS ENHANCE WITH
ARROWHEAD
Hippurate hydrolysis test: test to detect Hippurate hydrolase or hippuricase enzyme produced by S.
agalactiae
In this test Sodium hippurate is hydrolyzed into benzoic acid and glycine.
To detect benzoic acid: ferric chloride (not performed)
To detect gylcine use: Ninhydrin reagent (rapid)
Positive Result: DEVELOPMENT OF PURPLE COLOR
FOR ALPHA-HEMOLYTIC STREPTOCOCCI
USE FOR S. PEUMONIAE OR VIRIDAS
Neufeld quelling (+):
Bile solubility test
Required Media:
Reagent: Sodium Desoxycholate
Positive Results: Lysis of colonies
Negative Result: Intact Colonies, Turbidity or partial clearing
Optochin Disk Test / Taxo P
Chemical Name: Ethylhydrocupreine hydrochloride
Positive: S. peumoniae
Negative → BILE ESCULN → PYR
6mm disk = zone of inhibition should be 10mm to be considered SUSCEPTIBLE
10mm disk = zone inhibition should be 14mm to be considered SUSCEPTIBLE
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FOR GAMMA HEMOLYTIC STREPTOCOCCI
Screening test
Media: BILE ESCULIN MEDIA
Indicator: FERRIC AMMONIUM CITRATE
Positive: BLACKENING OF MEDIA
Differential test: PYR, PENICILLIN, SALT TOLERANCE TEST - 6.5%
Positive Result in Salt tolerance 6.5%: TURBIDITY
GROUP D NON-ENTEROCOCCI
ENTEROCOCCI
SALT TOLERANCE - 6.5%
PYR TEST PENICILLIN
NACL
GROUP D NON ENTERO - POSITIVE SUSCEPTIBLE
ENTEROCOCI + NEGATIVE RESISTANT
TO PREPARE A SALT TOLERANCE TEST
YOU NEED TO PREPARE BHI ( BRAIN HEART AGAR) + 6.5% SALT.
TUBE GROWTH: TURBID
PLATE GROWTH: COLONIES
TAKE NOTE OF THE LANCEFIELD CLASSIFICATION
💡 GROUP C, F, G = BETA
GROUP D = GAMMA
UNDER GROUP D
GROUP D NON ENTERO
GROUP D ENTEROCCI (IT IS NOW A SEPARATE GROUP)
Staphylococcus aureus
Characteristics
GM + cocci grape-like in clusters
Butyrous (battery-looking)
Colonies with an odor similar to an “ OLD SOCKS ON MSA "
BAP: Beta-hemolytic
MSA: PROLONG INCUBATION, Odor: OLD SOCKS ON MSA "
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Biochemical test
CAT +
COAG +
MF+
DNASE+
VP+
PYR -
Diseases
FOOD POISINING
TOXIC SHOCK
CALLED SKIN SYNDROME
RITTER DISEASE
VIRULENCE FACTOR
MAJOR VIRULENCE FACTOR: COAGULASE
CATALASE: NOT A VIRULENCE FACOTR.
Protein A: Prevents Phagocytosis
MRSA/ORSA:
Strain of S. aureus is resistant to Methicillin, Nefcillin and Oxacillin
Resistant of S. aureus to penicillinase is due to the PBP2a encoded by:
mec A Gene
Treatment for MRSA: Vancomycin (glycopeptide)
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💡 Why S. aureus resistant to Methicillin?
Because of it’s PBP2A (penicillin binding protein 2) coded by mec A gene
Treatment for MRSA: Vancomycin
Detection Method:
Questions? how we can detect MRSA+ S
Use of CHOM AGAR
MRSA Positive Result: ROSE OR MAUVE COLONY COLOR
NON-MRSA Negative Result: No colonies or bluish colonies
CEFOXOTIN DISK DIFFUSION TEST- induces expression of PBP2A
GOLD STANDARD FOR MRSA DETECTION
PCR TEST: DETECTS FOR mec A Gene
COAGULASE-NEGATIVE STAPHYLOCOCCI
S. EPIDERMIDIS
Pathogenesis: Cause PROSTHETIC HEART VALVE INFECTION
VIRULENCE FACTOR: SLIME PRODUCTION OR BIOFILM FORMATION
Enhance attachment of organism to plastic catheters
S. SAPHROPHTICUS
Pathogenesis MOST COMMON UTI IN YOUNG WOMEN
SMITH & BROWN CLASSIFICATION HEMOLYSIS ON BAP
ALPHA: Incomplete or partial
GREENING OF AGAR
BETA: Complete Hemolysis
CLEAR ZONES AROUND THE HEMOLYSIS
GAMMA: Unable to cause Hemolysis
NO PRESENCE OF HEMOLYSIS AND GREENING OF AGAR
Lancefield classification
Group A: S. pyogenes
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Characteristics
Gram + cocci, PYR +, Bacitracin-Susceptible, BETA HEMOLYTIC
Causes
Bacterial pharyngitis/strep throat
Pyoderma infection: Erysipelas
Scarlet fever: STRAWBERRY TONG: Pharyngitis with RASHES
Necrotizing Fasciitis: Rapidly progressing rapid skin infection
POST STREPTOCOCCAL SEQUELAE: ACUTE GLUMEROLONEPHRITIS & RHEUMATIC FEVER
Media
Selective: PEA Agar
Medium of choice: SBA
VIRULENCE FACTORS
M Protein: Prevents phagocytosis
emm gene codes for m protein
Major virulence factor
Protein F: Attachment of epithelial cells
Streptokinase: Dissolution of Clot
Streptolysin O
Streptolysin O:
Causes Hemolysis on BAP
Antigenic
Oxygen labile
Streptolysin S
Non antigen
Oxygen Stable
Can cause Hemolysis when incubate aerobically
Erythrogenic or pyrogenic toxins
responsible for the rashes of scarlet fever
2. STAPHYOCCI AND MICROCOCCI 9
Dick’s if the person is at risk at developing scarlet fever
Group B: S agalactiae
Normal flora of female genital tract or lower GIT: causes Septicemia
Number 1 cause of Bacterial Meningitis
can cause Postpartum endometriosis in adults
TODD HEWIT BROWTH MEDIA used to detect genital carriage of group b during pregnancy
LIM broth for
2. STAPHYOCCI AND MICROCOCCI 10
💡 Enterococci
Can be an agent of hospital-acquired UTI
💡 Non Enterococci
S. bovis and S. equinos = same species
Note: Bovis is not a valid species anymore
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