MODULE 2 GRAM-POSITIVE COCCI

MODULE 2 GRAM-POSITIVE COCCI

Family Micrococcaceae
Classification Family Micrococcaceae GENUS GENUS GENUS GENUS Staphylococcus Micrococcus Stomatococcus Planococcus

GRAM-POSITIVE COCCI MICROCOCCACEAE STREPTOCOCCACEAE STAPHYLOCOCCUS
S. aureus S. epidermidis S. saprophyticus STREPTOCOCCACEAE STREPTOCOCCUS S. pyogenes S. pneumoniae S. agalactiae E. faecalis S. mutans S. mitis viridans S. sanguis

STAPHYLOCOCCUS Two groups of staphylococci based on their ability to clot plasma(coagulate) 1) Coagulase-positive: S. aureus 2) Coagulase-negative staphylococci may be: a) Reported as such or b) Identified to species through additional testing. c) S. epidermidis & S. saprophyticus are CNS.

STAPHYLOCOCCUS COMMENSALS: Skin Mucous membranes

Natural habitat Staphylococci – resistant to dry & high salt conditions. Opportunistic pathogens Present in 20 – 80% on anterior nares.

STAPHYLOCOCCUS SYNDROMES

STAPHYLOCOCCUS S. saprophyticus S. aureus Urinary Tract infections
S. epidermidis Prosthetic devices Bacteremia Bone infections S. aureus Skin infections Respiratory tract infections Nosocomial infections Food Poisoning Bacteremia

STAPHYLOCOCCUS Staphylococcal scalded skin syndrome

Scalded skin syndrome:
Staphylococcal toxins – responsible for this condition. Usually occur in neonates & infants Due to exfolitin toxins: Skin sloughs

STAPHYLOCOCCUS Toxic shock syndrome

Toxic shock syndrome (TSS)
Symptoms: high fever, hypotension, confusion, diffuse rash and acute renal failure. Most early cases were assocaited with menstruating women – tampons. Now occurs in men & women – complication of staph infections.

STAPHYLOCOCCUS Pustular impetigo – superficial skin infection

superficial skin infection
STAPHYLOCOCCUS Bullous impetigo superficial skin infection

STAPHYLOCOCCUS Boil

(infection around the hair follicles)
Folliculitis (infection around the hair follicles)

Wound infections

STAPHYLOCOCCUS AUREUS
Predisposing factors Immunodeficiencies Skin injuries Prosthetic devices Other infections (eg. HIV) Chronic diseases (eg. Alcoholism, heart disease) Prophylactic / therapeutic antibiotics

STAPHYLOCOCCUS PROSTHETIC DEVICES

STAPHYLOCOCCUS saprophyticus
Causes acute urinary tract infections Causative agent of pyelonephritis, urethritis & catheter-associated urinary tract infections.

STAPHYLOCOCCUS epidermidis
Together with S. aureus – often isolated in infections associated with prosthetic devices: -> Joint prostheses, cardiovascular devices, artificial heart valves. -> IV catheters

Conditions include: Endocarditis CSF shunt infections Peritoneal dialysis catheter-associated peritonitis Bacteraemia Osteomyelitis Vascular graft infections Urinary tract infections

General characteristics
STAPHYLOCOCCUS General characteristics Morphology : Gram positive Cocci (clusters) Catalase positive No spores

STAPHYLOCOCCUS General characteristics Physiological: Facultative anaerobes Non-motile Usually non-capsulate Grow on routine media

STAPHYLOCOCCUS S aureus S epidermidis

Culture: Optimum temperature 35°C - 37°C. Growth on blood agar, nutrient agar, mannitol salt agar, MacConkey agar without crystal violet. Within 24hrs 1 – 3 mm diameter colonies (also dwarf colonies).

Colonial morphology 1) Blood agar a) Opaque, smooth, low convex, glistering with entire edges & butyrous (butter- like)consistency. b) S. aureus: Often beta-haemolytic(due to presence of b-haemolysins) & yellow-gold pigmented c) CNS: Usually non-haemolytic and white colonies.

d) Older colonies of S. aureus will appear translucent & sticky
d) Older colonies of S. aureus will appear translucent & sticky. Capsulated strains of S. aureus will form large convex glistening colonies with cream to buff to gold pigmentation, which when slimy, will run over the surface of a tilted dish. Staphylococcus aureus colonies showing haemolysis on blood agar

2) Mannitol salt agar (MSA)
a) Selective & differential media b) High salt concentration (7.5%) inhibits most gram-negative and many gram-positive organisms. c) If mannitol fermented, acid produced d) pH indicator (phenol red) turns from red to yellow e) S. aureus i) Ferments mannitol ii) Colonies are yellow with yellow zones

Staphylococcus epidermidis ATCC™ 12228 Uninoculated Plate Staphylococcus aureus ATCC™ 25923

f) Coagulase-negative staphylococci & micrococci
i) Grow but most do not ferment mannitol ii) Red colonies with red zones d) Catalase: Positive

Identification Catalase test: a) Introduction 1) Differentiates staphylococci (+) from streptococci (neg) 2) Important in the identification of many other organisms b) Principle: Catalase, an enzyme, breaks down hydrogen peroxide to water and oxygen with bubbles formed by the released oxygen. H2O2 -> H2O + O2 (bubbles)

c) Procedure 1) Place a drop of 3% H2O2
c) Procedure 1) Place a drop of 3% H2O2. 2) Place colony of test organism into the H2O2. d) Interpretation 1) POSITIVE: immediate production of bubbles 2) NEGATIVE: No bubbles

e) False-positive reaction
1) If test performed with an iron loop 2) Carefully remove colonies growing on BA (RBC have small amount of catalase) 3) Pseudocatalase: Some bacteria (e.g. enterococci) produce pseudocatalases which decompose H2O2. These false-positive results may be avoided by reading the test within 20 to 30 seconds.

NOTE: A Control organism should be tested as hydrogen peroxide is unstable Hydrogen peroxide is unstable and should be stored in a spark-proof fridge. Avoid any undue exposure to light. The enzyme is present in viable cultures only. Do not perform on cultures over 24 hours old. Older cultures may give false-negative reactions.

Coagulase a) Introduction 1) Enzyme has 2 forms (bound and free) 2) Converts fibrinogen to fibrin 3) Reagent = plasma

b) Slide coagulase (bound coagulase, clumpling factor) test 1) Principle a) Bound coagulase attached to the cell wall b) When S. aureus is mixed with plasma, visible clumps of cells appear as fibrin strands are formed.

2) Procedure a) Use microscope slide b) Mix organism in saline – heavy smooth suspension c) Mix in a drop of plasma with applicator stick 3) Interpretation a) Positive: Appearance of clumps in 10 – 15 seconds. Report organism as S. aureus

b) Negative: No clumping
c) Invalid: autoagglutinates (clumps in saline) 4) Perform tube test a) On slide coagulase-negative isolates since not all S. aureus strains are slide coagulase positive b) On strains which autoagglutinate (formed clumps in saline with slide coagulase)

c) Tube coagulase test 1) Principle a) Extracellular (free) coagulase forms complex with coagulase- reacting factor (CRF), in plasma b) Complex converts fibrinogen to fibrin c) Tube coagulase test, test for both bound and free coagulase. 2) Procedure a) Test organism emulsified in plasma in a tube b) Tube incubated at 35°C for 4 hours.

3) Interpretation a) Gently tilt tube to detect clot formation (+ result) b) Re-examine negative tubes after overnight incubation at room temperature d) Sources of error 1) Read slide test within seconds to avoid false (+) results 2) False (+) or false (-) results with non-sterile plasma

3). False (-) tube tests may occur with S
3) False (-) tube tests may occur with S. aureus strains that lyse clots after prolonged incubation 4) Methicillin-Resistant S. aureus (MRSA): Often slide coagulase negative and tube coagulase positive 5) Colonies from media with a high salt concentration (MSA) more likely to autoagglutinate. Best to use colonies from BA.

Latex agglutination (LA) test
a) Many clinical laboratories replaced slide and tube coagulase test with commercial LA test kits b) Detect clumping factor and protein A, a S. aureus cell wall antigen c) In addition, some kits also detect capsular polysaccharides d) MRSA may occasionally give false negative results

Thermonuclease test (Heat-stable DNAse)
a) Principle 1) Deoxyribonuclease (DNAse), an enzyme, cleaves deoxyribonuclease acid (DNA) into nucleotide subunits 2) S. aureus produces a heat tolerant DNAse b) Procedure for detecting presence of DNAse on a DNAse agar 1) Place a colony on the DNAse agar 2) Incubate overnight at 35°C. 3) Following day flood the agar with 1N HCL.

c) Interpretation: :Clear zone around organism growth indicates the presence of DNAse,therefore (+) : No clear zone - negative

Novobiocin susceptibility
a) Perform on Coagulase negative staph from urine cultures; presumptively identification for S. saprophyticus b) Principle 1) Agar plate inoculated from confluent growth with test organism 2) Paper disk with novobiocin (an antibiotic) place on agar 3) Novobiocin diffuses into the agar and produces a gradient of changing drug concentrations.

4). Check if test organism is inhibited by
4) Check if test organism is inhibited by drug, a zone of no growth (zone of inhibition) occurs around the disk c) Procedure 1) Broth suspension (0.5 McFarland turbidity standard) 2) Test MO (micro-organism) swabbed onto agar plate (Mueller-Hinton) 3) Place paper disk with 5ug novobiocin onto inoculated area 4) Incubate plate for hours at 35°C

e) Souce of error: Rarely other staph species resistant
d) Interpretation 1) Susceptible: Zone of inhibition =/> 16mm in diameter 2) Resistant: Zone < 16mm (presumptive S. saprophyticus) e) Souce of error: Rarely other staph species resistant

Other identification tests
a) Commercially prepared test – used to identify staphylococci to the species level b) Molecular and genetic techniques – used to identify, epidemiologic typing, and characterization of S. aureus (ID & characterization of coagulase negative staphylococci) c) Rapid tests for detection of MRSA 1) The mecA gene produces a penicillin- binding protein (PBP 2’ or PBP 2a) that is found in MRSA strains

2). Technologies available for detecting
2) Technologies available for detecting PBP 2’ include PCR, fluorescence, and latex agglutination as well as chromogentic agars.

Phosphatase Production
STAPHYLOCOCCUS TEST S.aureus S. epi S. sap Catalase + Coagulase - Haemolysin   Novobicin Resist S R Bacitracin Resist Phosphatase Production Mannitol Ferment Protein IDENTIFICATION

S. aureus Virulence factors

Cell-surface proteins expressed on surface of staphylococci & proteins secreted in environment = enable organism to overcome body’s immune system, invade & colonise tissue. Therefore cause infections. These proteins = virulence factors.

S. aureus Adherence factors: Virulence factors Coagulase
clumping factor(surface-associated protein) React with fibrinogen  fibrin = aggregation of S. aureus Virulence factors Promotes attachment of S. aureus to blood clots & traumatized tissues Detected by clot formation when adding undiluted plasma to saline suspension of organism =

cell-free coagulase + globulin plasma factor 
cell-free coagulase + globulin plasma factor  staphylothrombin Surface adhesin proteins Receptors for fibronectin, laminin collagen – these receptors promote bacterial attachment Lipases – hydrolyses lipids Enable organism to survive in sebaceous areas of body & enable organism to invade superficial skin areas.

S. aureus Evading the immune system: Virulence factors Protein A
binds IgG - This inhibit opsonisation, phagocytosis & clearance of organism from system Polysaccharide A microcapsule inhibits phagocytosis Coagulase Coagulase + globulin plasma factor = staphylothrombin – formation of fibrin layer around abscess protects from phagocytosis Virulence factors

S. aureus Invasion & spread: Virulence factors Hyaluronidase
hydrolyses hyaluronic acid Lipases hydrolyses lipids DNAse hydrolyses DNA Fibrinolysin (staphylokinase) Dissolves fibrin clots Virulence factors

S. aureus Damage of cells: Virulence factors Cytolytic toxins:
, , ,  & leukocidin Enterotoxins: Types A, B, C, D, E, G, H & I Heat stable Epidermolytic Toxins: Type A (heat stable) Type B (heat labile) Toxic shock syndrome toxin Virulence factors

S. aureus Others: Virulence factors Penicillinase
antibiotic resistance Catalase breakdown of hydrogen peroxide Virulence factors

STAPHYLOCOCCUS S aureus IDENTIFICATION

STAPHYLOCOCCUS IDENTIFICATION S epidermidis

STAPHYLOCOCCUS IDENTIFICATION Culture:
BA, MacConkey without Crystal Violet °C Aerobic & anaerobic Morphology of colonies: S. aureus: yellow (0.5 – 1 μm in size) S. epidermidis & S. saprophyticus: white Haemolysis: S. aureus: -haemolysis (usually) S. epidermidis & S. saprophyticus: normally gamma IDENTIFICATION

STAPHYLOCOCCUS S epidermidis S saprophyticus IDENTIFICATION S aureus

STAPHYLOCOCCUS IDENTIFICATION

STAPHYLOCOCCUS Mannitol Salt Agar IDENTIFICATION

STAPHYLOCOCCUS Catalase Test IDENTIFICATION H2O H2O + O2 Catalase

STAPHYLOCOCCUS Coagulase Test IDENTIFICATION Tube coagulase test
Slide coagulase test IDENTIFICATION Tube coagulase test

STAPHYLOCOCCUS IDENTIFICATION DNAse test Positive Negative S aureus
S saprophyticus

STAPHYLOCOCCUS IDENTIFICATION Novobicin sensitivity S saprophyticus
S epidermidis

STAPHYLOCOCCUS Staphylokinase test IDENTIFICATION Negative Positive

STAPHYLOCOCCUS Latex agglutination test IDENTIFICATION

STAPHYLOCOCCUS THERAPY Penicillin & derivatives
Also aminoglycosides, erythromcyin Drug resistant strains MRSA Vancomycin THERAPY

Coagulase-negative staphylococci a)
Coagulase-negative staphylococci a) Once considered to be nonpathogenic b) Most infections in 1) Immunocompromised patients 2) Those with indwelling medical device (IV catheters, joint prostheses, cardiovascular devices, artificial heart valves) c) S. epidermidis: Most commonly isolated species. Conditions include: Endocarditis, CSF shunt infections, peritoneal dialysis catheter- associated peritonitis, bacteremia, osteomyelitis, vascular graft infections and urinary tract infections.

d)S. saprohyticus: Urinary tract infections, especially in young healthy, sexually active women. Could also be the causative agent of pyelonephritis, urethritis & catheter assocaited urinary tract infections.

STAPHYLOCOCCAL WOUND SEPSIS
VIRULENCE FACTORS: (pg. 9) Coagulase Haemolysins Hyaluronidase Lipases Catalase Protein A Cytolytic toxins

STAPHYLOCOCCAL WOUND SEPSIS
SYMPTOMS: Oedema, erythema, pain, pus TREATMENT: Remove foreign body Drain the wound Clean & disinfect Only use antibiotics if malaise & fever develops

STAPHYLOCOCCAL FOOD POISONING
INTOXICATION (pg. 10)  heat stable enterotoxins –neurotoxic room temp/warmer temp) SYMPTOMS Vomiting diarrhoea (watery stools) nausea abdominal pain & cramps Onset: 1 – 8 hrs

STAPHYLOCOCCAL FOOD POISONING
TREATMENT: No antibiotics (toxic effect NOT organism itself) Fluid replacement CONTROL: Correct storage of food Good Personal Hygiene

Streptococci in general
Streptococci in general - Characteristics 1. Gram stain: Gram positive cocci that tend to form chains 2. Culture conditions: a) Facultatively anaerobic b) Grow on routine laboratory (BA,CHOC) 3. Catalase: Negative 4.Common names: beta strep, alpha strep and pneumococci

STREPTOCOCCUS Classification: Haemolysis eg -haemolytic strep
Serology – Lancefield antigens Biochemistry

STREPTOCOCCUS Classification: Haemolysis eg -haemolytic strep Streptolysin O – absence of oxygen Streptolysin S – presence of oxygen -> Stab, cut plate or incubate anaerobically to improve detection -> If MO produces only SLO, may miss haemolysis if O2 present -> MO with both toxins have enhanced haemolysis in stab or cut

STREPTOCOCCUS LANCEFIELD ANTIGENS: Of the cell wall
Either polysaccharides Group A: S. pyogenes Group B: S. agalactiae or lipotechoic acids: Group D: E. faecalis

STREPTOCOCCUS Serotyping LANCEFIELD ANTIGENS: …
Lacking in certain species: S. pneumoniae Viridans streptococci Serotyping

STREPTOCOCCUS COMMENSALS: Skin eg S. pyogenes
Respiratory tract eg S. pyogenes S. pneumoniae Intestinal tract eg E. faecalis

STREPTOCOCCUS General characteristics Morphology: Gram positive Cocci Physiology: Facultative anaerobes Non-motile Biochemical: Catalase negative

STREPTOCOCCUS SYNDROMES S. pyogenes

STREPTOCOCCUS SYNDROMES Scarlet fever- S.pyogenes Strawberry tongue
Pastias line

STREPTOCOCCUS SYNDROMES Erysipelas - S. pyogenes

STREPTOCOCCUS SYNDROMES Necrotizing fasciitis - S. pyogenes

STREPTOCOCCUS SYNDROMES S. pneumoniae S. pyogenes

STREPTOCOCCUS SYNDROMES E. faecalis: UTI wounds Endocarditis
Septicemia S. viridans: Dental caries Endocarditis S. agalactiae: neonatal meningitis perinatal infections endocarditis SYNDROMES S. pyogenes

S. pyogenes IDENTIFICATION Morphology: Arrangement: chains
Encapsulated/unencapsulated Cell wall antigens: Lancefield group A M protein T protein R protein IDENTIFICATION

S. pyogenes Morphology: IDENTIFICATION Knee tap fluid

 Aka -haemolytic streptococci Group A
S. pyogenes Culture: -haemolysis IDENTIFICATION  Aka -haemolytic streptococci Group A

S. Pyogenes Virulence factors

S. Pyogenes Virulence factors Adhesion factors
Lipotechoic acid – mediates adherence to epithelial cells. M protein – protect against phagocytosis & complement activity. F protein – Adherence to epi’s of pharynx & skin. Invasion M protein(Absent – non-infectious) F protein Virulence factors

S. Pyogenes Virulence factors Spread Streptokinases A & B
plasminogen  plasmin  fibrin & fibrinogen  lysis of clots – spread of org. DNAses – facilitate spread. Hyaluronidase – promotes spread. Virulence factors

S. Pyogenes Virulence factors Evasion
Capsule – same type of hyaluronic acid as in mammalian connective tissue M protein – binds factor H (complement protein) M-like proteins – binds IgM & IgG Virulence factors

S. pyogenes Virulence factors Toxins Streptococcal pyrogenic exotoxins
Streptolysin S Streptolysin O Virulence factors

S. pyogenes Virulence factors Toxins Streptococcal pyrogenic exotoxins
Produced by lysogenic strains 3 heat labile toxins:A, B, C Toxins enhance delayed hypersensitivity Enhance host cell susceptibility to endotoxins Toxins responsible for rash (Scarlet fever) superantigens  activation of Tcells that bind to major histocompatible complex molecules -> therefore massive release of cytokines  shock & organ failure  rash in Scarlet fever S. pyogenes Virulence factors

S. pyogenes Virulence factors Toxins
Streptococcal pyrogenic exotoxins … S. pyogenes Virulence factors Superantigens

S. pyogenes Virulence factors Toxins Streptolysins – cell lysis
non-immunogenic oxygen stable (aerobic) Streptolysin O immunogenic (anti-ASO test) oxygen-labile (anaerobic) Virulence factors

S. pyogenes Toxins immunogenic (anti-ASO test) Virulence factors

Diseases 1. Suppurative (pus forming) and toxic disease
a) Pharyngitis (strep throat), scarlet fever, bacteremia b) Skin infections/pyoderma (eg. Erysipelas, cellulitis & impetigo) c) Streptococcal toxic shock syndrome 2. Nonsuppurative: Rheumatic fever & Acute Glomerulonephritis

Antigen detection – pharyngitis:
1. Throat cultures take hours of incubation 2. Group A Streptococci antigen test results in minutes 3. Methodologies: Latex agglutination, coagglutination & ELISA 4. Specificity – high, Sensitivity 60% to 95% 5.Confirm negative antigen test with culture

Identification methods
1.Bacitracin (Ba disk) susceptibility (disk identifies group A strep) a)Priniciple: Low concentration bacitracin (antibiotic) inhibits Group A Streptococci b)Procedure 1)Inoculate BA for confluent growth 2)Paper disk (0.04 units of bacitracin) placed onto plate 3)Incubate overnight

c) Interpretation: Susceptible (S) = Any zone of inhibition d) Reporting results: Bacitracin susceptible beta-haemolytic strep may be reported as “Presumptive group A streptococci” e) Sources of error 1) Test only beta-haemolytic strep; some alpha-haemolytic strep(S) 2) False (+) with some group B,C & G streptococci 3) Group A Streptococci rarely bacitracin resistant

2. PYR (L-pyrrolidonyl-beta- naphthylamide) hydrolysis test
a) Principle L-pyroglutamylaminopeptidase L-pyrrolidonyl-B-naphtylamine  Free B-naphthylamine + N,N-dimethylaminocinnamaldehyde -> Red colour

b) Procedure 1) Place test MO onto disk with PYR, wait a few minutes 2) Add developing reagent c) Interpretation 1) Positive: Red 2) Negative: No colour change d) Source of error 1) Variety of other MO PYR (+) 2) Interpret in conjunction with MO’s gram stain, colonial morphology, type of haemolysis & catalase reaction.

3. Serogrouping: Commercial kits; eg. Latex agglutination

S. pyogenes Treatment penicillin erythromycin Virulence factors

S. pneumoniae IDENTIFICATION AKA: -haemolytic strep Morphology:
lancet shape diplococci capsule no lancefield antigens 2 forms techoic acid: C polysaccaride (binds to CRP) F antigen IDENTIFICATION

S. pneumoniae Morphology: IDENTIFICATION

S. pneumoniae Morphology: IDENTIFICATION S. pneumoniae in sputum

S. pneumoniae Morphology: IDENTIFICATION S. pneumoniae

S. pneumoniae Morphology: IDENTIFICATION

S. pneumoniae IDENTIFICATION Culture:-haemolysis (aerobic with CO2)
-haemolysis (anaerobic) IDENTIFICATION

S. pneumoniae IDENTIFICATION Culture: draughtsman colonies
- amidase (autolysin) IDENTIFICATION

S. pneumoniae Virulence factors Adhesion factors:
surface protein adhesins Spread Neuramidase – by attaching the glycoproteins & glycoplipids of host cells. Virulence factors

S. pneumoniae Virulence factors Evasion IgA proteases
Capsule - antiphagocytic Phosphorylcholine Soluble capsular polysaccharides Tissue damage Pneumolysin Techoic acid Hydrogen peroxide Virulence factors

S. pneumoniae TREATMENT Penicillin Cephalosporins Erythromycin
Cotrimaoxazole TREATMENT

S. agalactiae IDENTIFICATION Morphology Chains Capsule
Beta/gamma-Haemolysis Lancefield group B C protein IDENTIFICATION

S. agalactiae IDENTIFICATION
Culture narrow zone of -haemolysis IDENTIFICATION Colonial morphology: Large, flat, creamy, small zone of beta-haemolysis

Normal habitat: Genitourinary tract Diseases 1
Normal habitat: Genitourinary tract Diseases 1. Neonates (Group B strep cause serious infections in Babies) a) Neonatal sepsis & meningitis + others b) Infants acquire MO during birth, disease occurs in a few c) CDC recommends screening all pregnant women for vaginal & rectal GBS colonization 2. Other infections: Variety in adults, especially in immune-compromised

Antigen detection tests
1. Commercial kits; LA antigen (CSF, serum, urine and vaginal secretions) 2. False (+) results in urine if skin or peri-rectal area colonized, rare with CSF

Identification 1. CAMP test (developers: Christie, Atkins & Munch-Peterson) a) Principle 1. Some S. aureus produce “beta-lysin” (a toxin) 2. Group B Streptococci produce “CAMP factor” 3. CAMP factor + beta-lysin = arrowhead-shaped are of enhanced haemolysis.

b) Procedure 1) Streak S. aureus down the middle of a BA. 2) Streak test MO perpendicular to staph streak, incubate. c) Interpretation 1) Positive: Arrowhead-shaped haemolysis (Group B Streptococci) 2) Negative: No enhancement (most other streptococci)

d) Sources of error 1) False (+): Some Group A Streptococci (10%) CAMP positive 2) False (-): About 2% of Group B StreptococcimCAMP negative 3) Not all S. aureus strains produce beta- lysin 4) Run (+) & (-) controls each time test performed

2. Bacitracin disk test: Group B Streptococci are resistant.
3. Hippurate hydrolysis a) Priniciple hippuricase hippurate > sodium benzoate + glycine b) Sodium benzoate detection 1) Special broth + MO incubated overnight 2) Test supernatant by adding 7% ferric chloride

3) Positive: Precipitate after 10 minutes 4) Negative: Precipitate clears within 10 minutes Sodium benzoate + 7% Ferric chloride  precipitate

Glycine + ninhydrin  blue/purple colour
c) Glycine detection 1) Place disk with hippurate in tube with water 2) Add test MO, incubate for 2 hrs, add ninhydrin 3) Positive: Blue/purple colour after 10 minutes 4) Negative: No colour change Glycine + ninhydrin  blue/purple colour

d) Sources of error (SOE)
1) Sodium benzoate method: Reading too soon 2) Ninhydrin method: wrong media 3) Other MO hippurate (+);Interpret in conjunction with MO’s gram stain & colonial morphology 4. Serogrouping: Commercial kits available

S. agalactiae VIRULENCE FACTORS DNAses hyaluronidase neuramidase
proteases hippurase haemolysins VIRULENCE FACTORS

Viridans Streptococci
Group Species Anginosus S. anginosus, S. constellatus, S. intermedius Mitis S. mitis, S. pneumoniae, S. sanguis, S. parasanguis, S. gordonii, S. crista, S. Oralis Mutans S. mutans, S. sobrinus, S. cricetus, S. rattus, S. downei, S. macacae Salivarius S. salivarius, S. vestibularis, S. thermophilus Bovis S. bovis, S. alactolyticus, S. equines Ungrouped S. acidominimus, S. suis

Morphology: chains -ve for Lancefield antigens IDENTIFICATION

Culture:  &  haemolytic small colourless colonies IDENTIFICATION

Lancefield Group D Streptococci
Nomenclature a) Sterptococci with group D antigen divided into 2 subsets “enterococci” and “non-enterococcal group D streptococci” b) Enterococci now in own genus, Enterococcus Enterococci more resistant to antibiotics d) S.bovis is the non-enterococcal Group D streptococci usually found in humans

Morphology: Gram positive Cocci a or g haemolysis Arrangement: chains Cell wall antigens: Lancefield group D

Biochemical: Nutritional fastidious Catalase negative Normal habitat: Gastro-intestinal tract Diseases: Variety. S. bovis bateremia associated with colon cancer

Enterococcus faecalis
Morphology: Oval shape Short chains Group D Lancefield antigens IDENTIFICATION

Culture: large white colonies non-haemolytic IDENTIFICATION

Adhesion: Aggregation substance Enterococcal surface proteins Carbohydrate adhesins Tissue damage Cytolysin Pheremones Spread Gelatinase VIRULENCE FACTORS

esculetin + ferric ions  black colour
Identification: 1.Bile-esculin test a)Principle: 1)Determines ability to hydrolyze esculin if 40% bile present. 2) High bile concentration inhibits many organisms 3) Agar has ferric ions 40% bil esculin  esculetin + glucose esculetin + ferric ions  black colour

b) Procedure: Inoculate agar & incubate c) Interpretation: 1) Positive: Black (non-enterococcal Group D Streptococci & enterococci) 2) Negative: No colour change d) Sources of error: some viridans strep – faint black colour if heavy inoculum.

2. 6.5% salt tolerance test a) Principle: Determines MO’s ability to grow in 6.5% NaCl b) Procedure:Inoculate broth with 6.5% NaCl, incubate c) Interpretation: 1) Positive: Turbidity or acid pH 2) Negative: No growth or pH change

LAB ID OF STREPTOCOCCI Gram stain S mitis S pneumoniae E faecalis
S agalactiae S pyogenes

Viridans streptococci
LAB ID OF STREPTOCOCCI Culture: Blood agar: aerobic & anaerobic S pneumonaie E faecalis S agalactiae S pyogenes Viridans streptococci

LAB ID OF STREPTOCOCCI Biochemical ID: Catalase negative

LAB ID OF STREPTOCOCCI Biochemical ID: …
Confirmation of S. pneumoniae … 1. Bile solubility test positive negative

Confirmation of S. pneumoniae … 2. Optochin sensitivity Optochin sensitive

Confirmation of Group D Streptococci … 1. Hydrolysis of bile aesculin Positive

Positive for E. faecalis
LAB ID OF STREPTOCOCCI Biochemical ID: … Differentiate between species of Group D 1. Ability to grow in 6.5 % NaCl Negative Positive for E. faecalis

LAB ID OF STREPTOCOCCI ID: … Other groups of streptococci
1. Haemolysis pattern

 Viridans streptococci
LAB ID OF STREPTOCOCCI ID: … Other groups of streptococci 1. If  or -haemolytic  Viridans streptococci

1. If -haemolytic  bacitracin sensitivity

1. If -haemolytic  bacitracin sensitivity Group A (S. pyogenes)  bacitracin sensitive

1. If -haemolytic  L-pyrrolidonyl arylamidase test Group A (S. pyogenes)  positive

1. If -haemolytic  sodium hippurate test Group B (S. agalactiae)  positive

1. If -haemolytic  CAMP test Group B (S. agalactiae)  positive

LAB ID OF STREPTOCOCCI Serological confirmation: … S. pyogenes:
latex agglutination of Group A antigens antistreptolysin O test antiDNAse B antistreptokinase

LAB ID OF STREPTOCOCCI Serological confirmation: … S. pneumoniae:
quellung reaction

LAB ID OF STREPTOCOCCI Serological confirmation: … S. agalactiae:
latex agglutination – Group B antigens

LAB ID OF STREPTOCOCCI Serological confirmation: … E. faecalis:
latex agglutination – Group D antigens

MODULE 2 GRAM-POSITIVE COCCI

Similar presentations

Presentation on theme: "MODULE 2 GRAM-POSITIVE COCCI"— Presentation transcript:

Similar presentations

About project

Feedback

Log in

Auth with social network:

MODULE 2 GRAM-POSITIVE COCCI

Similar presentations

Presentation on theme: "MODULE 2 GRAM-POSITIVE COCCI"— Presentation transcript:

Similar presentations

About project

Feedback