Gram staining - Cocci-Microorganism PDF

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Lecture Summary of Microbiology in identification of Gram staining Cocci microorganism. Topic lectured by Sir Rodney Guillermo, RMT...

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BACTERIOLOGY PROPER Gram (+) cocci – morphologically round in shape – violet/purple Kingdom Family Genus

Monera Micrococcaceae Micrococcus Staphylococcus Stomatococcus Planococcus

Monera Streptococcaceae Streptococcus Enterococcus Aerococcus Leuconcoccus Gemella Pediococcus

Point of Difference: Morphology: Gram Stain Growth characteristics (5% SDA) Colonies Hemolysis Catalase test O/F test Modifies Oxidase Test Lystostaphin Furazolidone (100 ug disk) Bacitracin (0.04 ug disk)

Micrococcus Pairs, tetrads, irreg. clusters Obligate aerobe

Staphylococcus Clusters Facultative aerobe

Streptococcus Chains Facultative anaerobe

Small-medium Non-hemolytic

Medium-large (pinhead) Beta-hemolysis Non-hemolytic + Glucose fermenter (+) Glucose oxidizer (+) S R R

Small (pinpoint) Alpha, Beta, Gamma hemolysis -

+ Non-glucose fermenter (-) Glucose oxidizer (+) + R S S

V V Group A = S Others = R

Staphyloccocus spp. I. II.

Staphylococcus aureus – Golden-yellow colonies; pathogenic CONS (Coagulase Negative Staph)  S. aureus – lemon yellow colonies; chromogenic opportunistic pathogen  S. citreus – white pigment; chromogenic opportunistic pathogen  S. albus – white pigment  S. hyicus – animal pathogen  S. intermedius – animal pathogen  S. epidermidis – predominant normal flora on the skin; leading cause of Iantrogenic infection  S. saprophyticus – opportunistic pathogen; normal flora of skin; frequently cause UTI, abortion/miscarriages  S. lugdunensis – opportunistic pathogen; normal flora

Virulence Factors of S. aureus  Hemolysins (Alpha, Beta, Gamma) – pathogenic factor; destroys RBC  Toxins  Leucocidin – Panton-Valentine Factor  Exfoliatin – destroys cells and tissues; cause SSS  Enterotoxin A-D – cause food poisoning  TSST (Enterotoxin F) – causes Toxic Shock Syndome  Fibrolysin/Staphylokinase – responsible for lysin clot  Protein A – responsible for antiphagoytosis  Polysaccharide A – responsible for antiphagoytosis  Coagulase – localizes abcess  Hyaluronidase – degrades DNA components of tissue; spreading factor  Lipase – spreading factor

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DNAse – spreading factor Other enzymes produced which are used to identify S. aureus  Gelatinase  Thermonuclease

Diseases caused by S. aureus A. Local purulent infection  Boils  Wound infection  Furuncles (hair infection)  Carbuncles (hair infection)  Bullous impetigo – skin of burn patients  Sinusitis  Otitis media – middle ear infection. B. Sepsis C. Toxin-caused illness  Food poisoning  Toxic Shock Syndrome

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Laboratory Identification 1. Cultural characteristics  Pinhead, hemolytic pattern 2.

Gram stain  Circular (Clusters), violet

3.

Catalase test  H2O2 + Bacteria Catalase H2O + O2  Reagent: 3% H2O2  (+) result: effervescence

4.

Coagulase test (slide or tube method)  Best single pathogenicity test for staphylococcus  Slide: screening test; bound coagulase  Plasma  Tube: confirmatory test; unbound coagulase  0.5 mL plasma (rabbit’s plasma)  35ºC; initial time = 2 hours  4 hours incubation (if negative, continue incubation for 24 hours)  (+) formation of coagulum/clot

5.

Mannitol fermentation  Staphylococcus spp. Fermentative (Glucose)  Medium: Mannitol Salt Agar  (+) result: yellow [S. aureus]  (-) result: pink [CONS]

6.

DNAse test  DNAse Medium  Incubate at 25ºC for several hours  (+) zone of inhibition

7.

Gelatin Liquefaction/Hydrolysis Test  12% Gelatin Medium (Tube)

Penicillinase Catalase

Post-influenza pneumonia UTI Styles/Blebitis Pimples Meningitis

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Staphylococcal Scalded Skin Syndrome

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Stabbing method Incubate at 35ºC for 16-18 hours (+) Liquefaction  Refridgeration (30 mins)

Novobiocin susceptibility S. aureus + S R

Coagulase Novobiocin Polymyxin 9.

S. epidermidis S S

S. saprophyticus R S

O/F   

Closed method  add mineral oil to become a barrier for oxygen Open method Interpretation of results:  Closed = (+) yellow: Fermentative  Open = (+) yellow; Oxidative  If both (+) = Staphylococci  If Closed is (-) and Open is (+) = Micrococcaceae

10. Modified Oxidase Test  Active Chemical component Tetra-methyl-para-phenylene diamine dihydrochloride  Results:  (+) = blue to purple to black complex [Micrococci]  (-) = no color change [Streptococci]

Streptococcaceae – Streptococcus spp. General Characteristics:  Gram (+) cocci in chains  Facultative anaerobe  Catalase (-)

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Non-motile Pyogenic – disease is caused by pus Pinpoint colonies

Classification of Streptococcus spp. Basis of classification

Academic/Bergy Temperature

Smith and Brown Hemolysis

Pyogenic Viridance Entero Lactic

Alpha Beta Gamma

Members

I.

Academic/Bergy’s Classification 10ºC 37ºC Pyogenic – + Viridance – +

Entero Lactic II.

+ +

45ºC – +

+ +

+ –

Lancefield Serological (C-carb of cell wall) Groups A-G

Members Streptococcus pyogenes S. mutans S. salivarius S. mitis S. bovis S. anginosus Enterococcus faecalis Streptococcus faecalis

Smith and Brown Alpha

Beta

Gamma

Color around colonies Members

Clear S. equisimilis S. pyogenes S. agalactiae E. faealis *S. pneumoniae, S. pyogenes, S. agalactiae, E. faealis – true pathogens

III.

Green S. pneumoniae

Red Some Viridans group

Lancefield Members

Group A S. pyogenes

Group B S. agalactiae

Group C S. equisimilis S. equi S. dysogalactiae S. zoopidimidus

Species S. pyogenes

Hemolysis Beta

Group Antigen A

Common Name Group A Streptococci

S. agalactiae

Beta

B

Group B Streptococci

S. equisimilis

Beta

C

Group C Streptococci

E. faecalis

Beta

D

Enterococci

S. bovis S. equinus

Alpha/Gamma

D

Non-Enterococci

S. pneumoniae

Alpha

Pneumococcus/Diplococcus

Viridans

Alpha/Gamma

Viridans Streptococci

Laboratory Tests for Streptococcus spp. 1. Gram Stain  Gram (+) cocci in clusters 2. Cultural Characteristics  Pinpoint colonies 3. Catalase Test  (–) = no effervescence 4. Bacitracin Susceptibility (Taxo A)  Differentiates Group A Streptococci from other Streptococci  (+) = Group A Strep  (–) = Streptococci

Group D A. Enterococci  E. faecalis  E. faecium  E. avium  E. durans B. Non-Enterococci  S. bovis  S. equinus

Disease Pharyngitis Scarlet fever Acute Glomerulnephritis Rheumatic Fever Pyodema (Impetigo contagis, Erysepelas & St. Anthony’s Fire) Neonatal sepsis (blood) Meningitis (CSF) Pneumonia (Throat Swab) Puereperal (Vaginal Swab of mother) Pharyngitis Pyogenic infection UTI Wound Infection Bacteremia Endocarditis UTI Wound Infection Endocarditis Lobar pneumonia Meningitis Bacteremia Endocarditis

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CAMP Test (Christie Atkins Munch Petersen Test)  Test for Group B Streptococci  Known organism (S. aureus)  Unknown organism [Beta-hemolytic, Catalase (-), pinpoint, Bacitracin Resistant]  Group B Strep  S. agalactiae  (+) = arrowhead hemolysis [Group B Strep] or bowtie appearance [S. agalactiae] PYR Hydrolysis Test  (+) = Cherry Read/Pink  Test for Group A and Group B Streptococci  Substrate: L-pyrrolidonyl-beta-napthylamide  Reagent: p-dimethyl-aminocinnamaldehyde  Enzyme: pyrroglutamylamino peptidase or pyrrolidomylaryl amidase Hippurate Test  Same as CAMP test  Medium: Broth with hippurate  Reagent Indicator: Ninhydrin  Hippurate  Benzoic Acid +Glycine  Add indicator after incubation  (+) = Purple Bile Esculin Test  Group D (Enterococci/Non-Enterococci)  Differentiate Group D from Beta-hemolytic  Enzyme: esculinase  (+) = blackening of medium [Group D] Salt Tolerance Test (6.5% NaCl)  Further test from Bile Esculin Test  To identify if Enterococci  (+) = Clear/Transparent [Non-enterococci]  (–) = Turbid [Enterococci] SXT Susceptibility  R – Group A & B Strep  S – Other Strep spp. Serological Tests  Detect carbohydrate component of the cell wall of Streptococci  Name of test: Streptex  Principle: Antigen(from colonies)-Antibody(from Reagent) Reaction  Tube  Add diluent (PBS/NSS) [0.5mL]  Transfer bacteria  Transfer to test card labelled A-G  Add antibody  (+) = agglutination/clumping [Group A Strep]  (–) = no agglutination [S. agalactiae] Skin Test  Principle: antigen-antibody reaction  Schultz-Charlton Skin Test  Branching phenomenon  Patient must have rashes  To detect Scarlet Fever  Anti-erythrogenic toxin  (+) = disappearance of rashes [S. pyogenes]  (–) = [Rickettsia/Virus]  Dick’s Skin Test

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Test for previous infection Erythrogenic test Patient must not have rashes 2-3 days (+) = induration/wheale formation

Bacitracin SXT CAMP PYR Sodium Hippurate Bile Esculin 6.5 NaCl

A S R – + – – –

B R R + – – – –

C R S – – – – –

D R S – + – + Entero (+) Non-entero (–)

Virulence Factors of Streptococci spp.  M protein – part of cell wall; antiphagocytic  Capsule – principal virulence factor of Group B Streptococci; antiphagocytic  Hemolysin Streptolysin S Type of hemolysin Surface of RBC Oxygen Stable Antigenecity Non-antigenic 

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Toxin  Erythrogenic toxin – causes Scarlet Fever  Nephrogenic toxin - Acute Glomerulnephritis Streptokinase – spreading factors localized in the skin Hyaluronidase – spreading factors localized in the skin DNAse – spreading factors localized in the skin

Streptococcus pneumoniae 

Streptolysin O Subsurface Labile Antigenic

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Diplococcus /Pneumoco ccus

Gram (+) cocci in pairs

Laboratory Tests for Streptococcus pneumoniae 1. Gram Stain  Pairs/lancet shaped 2. Cultural Characteristics  Dome shaped 3. Optochin Test/Taxo P  Chemical composed of ethylhydrochaprin hydrochloride  S = Pneumococci  R = Viridans Streptococci  Do this test only when organism is Alpha hemolytic 4. Bile Solubility  For S. pneumoniae only  Sodium deoxycholate  Tube: 2% sodium deoxycholate  (+) = disappearance of colonies  Plate: 10% sodium deoxycholate

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Alpha hemolytic No Lancefield Calssification Dome-shaped colonies Green around colonies

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7. 8.

 (+) =Clear [S. pneumoniae]  (–) = Turbid [S. mitis] Inulin Fermentation  Only S. pneumoniae can ferment inulin (carbohydrate)  Indicator: Phenol Red  (+) = Yellow [S. pneumoniae ]  (–) = Red/Pink [Viridans Group] Capsular Swelling Reaction/Nueffeld Quellung Reaction  Methylene Blue + Antitoxin + Bacteria  (+) = Obvious cell wall [S. pneumoniae ]  (–) = No capsule [All other Alpha hemolytic Streptococci] Mouse Virulence Test  (+) = Death of test animal Francis Skin Test  Test for previous infection  Erythrogenic test  Patient must not have rashes  2-3 days  (+) = induration/wheale formation...