Microbiology + Mycology and Virology PDF

Preview

Summary

GRAM-POSITIVE COCCI Staphylococci Micrococci Streptococci Pneumococci Viridans Streptococci STAPHYLOCOCCI SPP. Gram (+) Cocci in GRAPE-LIKE CLUSTERS Able to grow in 7 – 10% NaCl (in a media) Resistant to .02 – .04 units of Bacitracin (aka Taxo A) Catalase test (+) S. AUREUS Normal flora of Anterior ...

Description

GRAM-POSITIVE COCCI • • • • •

1. 2. 3. 4. 5. 6.

Staphylococci Micrococci Streptococci Pneumococci Viridans Streptococci

STAPHYLOCOCCI SPP.

-

• • • •

Gram (+) Cocci in GRAPE-LIKE CLUSTERS Able to grow in 7.5 – 10% NaCl (in a media) Resistant to .02 – .04 units of Bacitracin (aka Taxo A) Catalase test (+)

• •

S. AUREUS Normal flora of Anterior nares and Nasopharynx Can cause TOXIN MEDIATED and NON-TOXIN MEDIATED diseases. TOXIN MEDIATED Food poisoning Toxic Shock Syndrome Scalded Skin Syndrome/ Ritter’s Disease

Coagulase

Beta lactamase

Hyaluronidase DNase/ Thermonuclease Beta Hemolysin

Enterotoxins A &B TSST-1 Exfoliatin Panton Valentine Leukocidin (PVL) Protein A STAPHYLOkinase

• • • o o o o o • o o

NON-TOXIN MEDIATED Boils, carbuncles, furuncles, cellulitis, wound infections (Skin infections) Stye (eye infection) -

VIRULENCE FACTORS Causes bacterial cell to agglutinate in plasma and converts fibrinogen into fibrin MARKER OF VIRULENCE Aka PENICILLINASE Responsible for S. aureus resistance to Penicillin. Treatment: Penicillinase-Resistant Drugs/ Methicillin Aka DURAN REYNALS FACTOR Invade tissues and is the spreading factor Degradation of DNA Decreases viscosity of exudates allowing more mobility. Aka SPHINGOMYELINASE C / HOT-COLD LYSIN Cause beta hemolysis Food poisoning

• •

BIOCHEMISTRY TEST CHARACTERISTICS Catalase (+) Coagulase (+) Mannitol Fermentation Test (+) DNase Test (+) Vogues Proskauer (+) PYR (-) CoNS – COAGULASE NEGATIVE STAPHYLOCOCCI Staphylococcus saprophyticus Staphylococcus epidermidis

Remember, NOVOBIOCIN SUSCEPTIBILITY TEST is used to differentiate CoNS.

Catalase Coagulase Mannitol Fermentation Hemolysis on BAP Novobiocin Susceptibility (5ug) DNAse Test Phosphatase Gelatinase Other

Virulence Factors

S. EPIDERMIS + -

S. SAPROPHYTICUS + -

GAMMA

GAMMA

Susceptible

Resistant

+ + Normal skin flora Causes UTI, stitch abscess Prosthetic Heart Valve infection

+ Most common cause of UTI (esp. in young women) Causes Pyelonephritis and cystitis in those with indwelling catheters

Slide production or Biofilm formation (Enhances adherence of organism to plastic catheters)

UNCERTAIN

Toxic Shock Syndrome Causes skin desquamation/ exfoliation in Scalded Skin Syndrome Destruction of WBCs

Prevents phAgocytosis Promotes clot lysis (dissolution of clot)

CULTURE CHARACTERISTICS Seen in GRAPE-LIKE CLUSTERS Uniform turbidity on broth On 5% BAP Zone of Hemolysis: BETA Hemolysis Size: Pinpoint, MEDIUM TO LARGE Color: Creamy Yellow “Buttery” Margins: Smooth entire Elevation: Slightly raised, translucent (+) growth on MSA, CAN MSA – Mannitol Salt Agar CNA – Columbia-Colistin Nalidixic Acid

1

BIOCHEMICAL TEST TEST CATALASE TEST

CEPHALOSPORINASE TEST

PRINCIPLE Detection of bacteria by the presence of enzyme Catalase that can convert Hydrogen peroxide to H2O and O2 Detection of Beta lactamase production

REAGENT 3% Hydrogen Peroxide

Use of CEFINASE DISK Substrate: Nitrocefin

RESULT POS: Bubbling / Effervescence

NOTES FALSE POS: use of colonies from BAP

NEG: No bubbles POS: Pink to Red Rapid Test NEG: Yellow FALSE POS: Citrated plasma SLIDE COAGULASE PROCEDURE (Loop fool organism + NSS + Rabbit’s plasma)

Ability to produce coagulase enzyme

COAGULASE TEST

Have two types: 1. Slide Coagulase – detects bound coagulase or Clumping Factor 2.

Rabbit’s plasma with EDTA

Fermentation of Mannitol and Production of Acid

Inhibitor: 7.5% Salts pH Indicator: Phenol Red

DNAse Test

NEG: No clot formation

Tube Coagulase – detects free coagulase

Use of MANNITOL SALT AGAR MANNITOL FERMENTATION TEST

POS: Clot Formation

Hydrolyze DNA based on the production of deoxyribonuclease

HCl Precipitation method: DNAse agar, 0.1 Normal HCl DYE Method: DNAse agar, Methyl green, Toluidine Blue (incorporate dye)

POS: YELLOW HALO around the colonies (acidic) + “OLD SOCK” Odor (For S. aureus) NEG: No growth (Pink agar; acidic) HCl Precip. method POS: Clearing of agar around the colonies DYE Method In Methyl Green POS: Clear zone In Toluidine Blue POS: Pink Zone NEG: No clearing/ change

TUBE COAGULASE Must be performed if Slide Coagulase is Negative. - Loop fool org. + NSS + Rabbit’s plasma - Incubate at 37 ° C for 4 hours - If neg., incubate again for 16 hrs. Report results (Total time = 20 hrs) Pos for S. aureus means: 1. Organism was able to ferment mannitol 2. Organism was able to tolerate increase con’c of NaCl

HCl PRECIPITATION PROCEDURE - Inoculate at DNAse agar - Incubate - Add 0.1 Normal HCl Moraxella & Serratia spp. are also POS for DNAse test. “SMS”

SUMMARY Catalase Coagulase Mannitol Fermentation Test DNAse Test Vogues Proskauer Phosphatase Gelatinase Novobiocin Susceptibility Test Hemolysis on BAP Marker of Virulence PYR

S. aureus

S. epidermidis

S. saprophyticus

+ + + + +

+ -

+ -

+ + Susceptible GAMMA Slide production or Biofilm formation

+ Resistant GAMMA

BETA Coagulase

uncertain

-

2

MICROCOCCI SPP. • • • •

STREPTOCOCCI SPP.

Gram (+) cocci in TETRADS and CUBOIDAL PACKETS Strict AEROBE GAMMA Hemolysis on BAP MODIFIED OXIDASE TEST (+) o Presumptive Test; can also be used in Neisseria spp. (Gram NEG) o Uses tetramethyl p-phenylenediamine dihydrochloride in DMSO (Dimethyl sulfoxide) § In Gram POS – use of 6% oxidase § In Gram NEG – use of 1% oxidase o RESULTS § POS – BLUE or PURPLE-BLUE color change within 2 minutes § NEG – No change in color

AERObic growth** ANAERObic growth Lysostaphin Susceptibility Modified Oxidase Test Bacitracin susceptibility Furazolidone/ Furoxone Susceptibility Catalase Test** Benzidine Test

STAPHYLOCOCCI ferment glucose produces acid WITH or WITHOUT AIR +

MICROCOCCI oxidize glucose produces acid ONLY in the PRESENCE OF AIR +

+

-

S

R

-

+

R

S

S

R

+ -

+ +

Gram (+) cocci in CHAINS, SPHERICAL to OVOID Catalase (-) LAP Test (+) Non-motile; CAPNOPHILIC (need increased CO2) Medium of Choice: Sheep’s Blood Agar (consider first the zone of hemolysis on BAP) o Enriched media that can detect hemolysis o Usually used in laboratory for initial identification • Selective Medium: Phenyl Ethyl Alcohol Agar (PEA) • SMITH & BROWN CLASSIFICATION o Classification of Streptococci spp. according to HEMOLYSIS PATTERN. ALPHA • Can cause INCOMPLETE/ PARTIAL HEMOLYTIC HEMOLYSIS on BAP • GREENING of Agar • Streptococcus pneumoniae, Viridans Streptococci – S. mitis & S. mutans BETA • Can cause COMPLETE HEMOLYSIS on BAP HEMOLYTIC • Produce CLEAR ZONES around the colonies • S. pyogenes (Group A Strep) & S. agalactiae (Group B Strep) GAMMA • DO NOT CAUSE HEMOLYSIS on BAP HEMOLYTIC • E. faecalis, S. bovis • • • • •

LANCEFIELD CLASSIFICATION o For further classification of Beta hemolytic Strep. o Most significant in identifying and classifying Beta hemolytic Strep o Based on the extraction of C carbohydrate from the Streptococcal Cell Wall. Group A Streptococcus pyogenes Beta hemolytic Streptococcus agalactiae Group B Beta hemolytic Considered as Normal flora of skin, Group C, F, G nasopharynx, GIT, Genital Tract Beta hemolytic •

3

TEST

MODIFIED OXIDASE TEST

LAP TEST (Leucine Amino Peptidase)

PRINCIPLE

Detection of oxidase enzyme

Detection of enzyme leucine amino peptidase

BIOCHEMICAL TEST REAGENT tetramethyl pphenylenediamine dihydrochloride in DMSO (Dimethyl sulfoxide) In Gram POS – use of 6% oxidase In Gram NEG – use of 1% oxidase Use of LAP disk Substrate: Leucine-betanaphthylamide Reagent: Cinnamaldehyde

PYR TEST (Pyrrolidonil Aminopeptidase)

Detection of Pyrrolidonil Aminopeptidase)

HIPPURATE HYDROLYSIS TEST

Ability of bacteria to hydrolyse Sodium Hippurate into glycine and benzoic acid (end product) by action of hippuricase/ Hippurate hydrolase enzyme

BILE ESCULIN TEST

SALT TOLERANCE TEST

OPTOCHIN SUCEPTIBILITY TEST

BILE SOLUBILITY TEST

NEUFELD QUELLUNG (CAPSULAR SWELLING TEST)

rapid detection of esculin hydrolysis in presence of bile Salt acts as a selective agent for bacteria and interferes with membrane permeability and osmotic equilibrium.

n-n dimethylaminocinnamaldehyde

Indicator: Ferric Chloride (to detect Benzoic acid)

RESULT POS – BLUE or PURPLEBLUE color change within 2 minutes. NEG – No change in color POS: RED NEG: No color or Slight yellow POS: RED NEG: No color change/ yellow

POS: PURPLE COLOR

Indicator: Ninhydrin (to detect Glycine)

NEG: No color change

Use of Bile Esculin Media (Tube media dispensed as SLANT)

POS: BLACKENING OF MEDIA

Indicator: Ferric ammonium citrate

NEG: No color change

Addition of 6.5% NaCl in Broth

Use of SBA

POS: GROWTH (turbidity) in tube media, (colonies) in plate media NEG: No growth POS: 14mm Zone of Inhibition around the disk

Disk diffusion Optochin reagent (on disk)

Sodium deoxycholate and sodium taurocholate selectively lyses

Anti-capsular antibodies present in the serum react with carbohydrate material of the pneumococcal capsule

Use of BAP In Media, add 10% Sodium desoxycholate Use of Broth Add 6% desoxycholate

Anti-serum for capsule

CYTOCHROME OXIDASE TEST

Detection of cytochrome oxidase enzyme

Tetramethyl-pphenylenediamine dihydrochloride

CHO UTILIZATION TEST

Fermentation of Carbohydrate

Use of Cystine Trypticase Agar (CTA) Indicator: Phenol Red

NEG: No zone of inhibition around the disk In MEDIA: POS: LYSIS OF COLONIES NEG: Intact colonies In BROTH: POS: CLEARING OF MEDIA NEG: turbidity POS: Presence of capsule “HALO” around dark blue stained cell NEG: no change/ halo/ swelling POS: PURPLE NEG: No color change POS: YELLOW (acidic) & AIR BUBBLE (Gas production)

NOTES PRESUMPTIVE & RAPID TEST to differentiate Staphylococcus to Micrococcus PRESUMPTIVE TEST for Catalase (-) gram (+) cocci (ID Streptococci spp.) PRESUMPTIVE IDENTIFICATION of group A beta-hemolytic Streptococci and Enterococci Two end-products can be detected: Benzoic acid & Ninhydrin Nowadays, Ninhydrin is more used for Rapid Test Screening test for Group D Non-Enterococci & Enterococci Inoculate à Incubate à see for a change in color

Add salt à inoculate à incubate à check results

Chemical name of Optochin: ethylhydrocupreine hydrochloride

Procedure: Inoculate à Incubate à Add reagent

S. pneumoniae is POS because it is encapsulated “CAPSULAR SWELLING” SCREENING TEST FOR NEISSERIA (1% rgnt without DMO)

Used in identifying spp. under Neisseria spp.

NEG: No color change on phenol red

4

• • •

• • •

S. PYOGENES Aka Group A Beta Hemolytic Streptococci in CHAINS Major throat pathogen Can cause: o Pharyngitis/ Strep throat o Necrotizing fasciitis – rapidly progressing skin infection; flesh-eating bacteria o ERYSIPELAS – skin infection § Erysipeloid – diff. agent (Erysipelothrix; gram POS RODS) o Scarlet fever – form of pharyngitis with rashes They may also develop AGN (Acute Glomerulonephritis) & Rheumatic fever 1st Sheep’s Blood Agar – Media for Initial isolation; Beta hemolysis Identification Tests o 2ndPYR (+) o 3rdBacitracin – SENSITIVE

M protein Streptolysin O

Streptolysin S

Protein F Erythrogenic Toxin/ Pyrogenic Toxin Others:

DICK’S TEST (DSSD)

SCHULTZ CHARLTON TEST or BLANCHE PHENOMENON

VIRULENCE FACTORS Prevents phagocytosis MAJOR VIRULENCE FACTOR Antigenic/ Immunogenic (stimulates release of anti-streptolysin O when introduced to the body) Oxygen labile (sensitive in oxygen) Can cause sub-surface hemolysis when incubated anaerobically Cause beta hemolysis Non-antigenic Oxygen stable Can cause SURFACE hemolysis when incubated aerobically) Cause beta hemolysis Promotes attachment (adherence) to epithelial cells

• • •

•

S. AGALACTIAE Aka Group B – Beta hemolytic Streptococci Considered as Normal Flora o But can cause POSTPARTUM ENDOMETRISIS Regarded as the #1 CAUSE OF NEONATAL MENINGITIS Other Meningitis caused by another agent: o < 5 y/o – Haemophilus influenzae o 5 to 29 y/o – Neisseria meningitidis o > 29 y/o – Streptococcus pneumoniae (Adult bacterial meningitis) o Elderly/ Immunocompromised – Listeria Identification Tests: o CAMP Test (+) o Hippurate Hydrolysis (+) o Bacitracin – RESISTANT

Principle

Required Media Known organism RESULTS

CAMP TEST hemolytic activity of the beta-hemolysin produced by most strains of S. aureus is enhanced by an extracellular protein produced by group B streptococci Both are B-hemolytic = “SYNERGISTIC EFFECT” BAP (Perpendicular Streak) Staphylococcus aureus POS: Enhanced hemolysis as shown by ARROW HEAD ZONE of Beta hemolysis NEG: No enhanced hemolysis

Responsible for rashes in scarlet fever Hyaluronidase (DRF) – spreading factor; enhances the ability of an organism to invade tissues. Streptokinase – promotes clot lysis; distortion of clot (both V.F. are also produced by S. aureus) Susceptibility test for Scarlet Fever • Detects immunity for Scarlet fever • Intradermal injection (Skin Test) • POS: Erythema; redness at the site of the injection à person is at risk of developing Scarlet Fever • Schick’s Test – often interchangeable with this test but is used for Diphtheria. Test to determine if rashes are due to scarlet fever or not • POS: fading of rashes due to injection of Anti-erythrogenic/ pyrogenic toxin • Neutralizes erythrogenic toxin released à fading of rashes (gradual disappearance

5

GROUP D – NON-ENTEROCOCCI • • •

S. bovis GROUP / S. gallolyticus group S. equinus, S. gallolyticus, S. infantarius, S. alactolyticus Encountered in blood cultures of patients with bacteremia, septicemia, & endocarditis

•

E. faecalis, E. faecium, E. durans, E. avium

ENTEROCOCCI

DIFFERENTIAL TESTS FOR ALPHA HEMOLYTIC STREPTOCOCCI S. PNEUMONIAE VIRIDANS Optochin S Susceptibility/ R Taxo P Bile Solubility Test + Neufeld Quellung +

Back then, both Enterococci and Non-Enterococci belonged to Group D. In today’s time, Enterococci is no longer part of Group D. • Both has GAMMA hemolytic pattern. • BILE ESCULIN TEST – Screening test for Group D NonEnterococci and Enterococci; both are POSITIVE

DIFFERENTIAL TEST FOR GROUP D – NON-ENTEROCOCCI AND ENTEROCOCCI NON-ENTEROCOCCI ENTEROCOCCI PYR TEST + Penicillin R Susceptibility S Test Salt Tolerance Test using 6.5 + NaCl

NUTRITIONALLY VARIANT STREP (NVS) • •

GRAM NEGATIVE COCCI

ALPHA HEMOLYTIC STREPTOCOCCI • • •

• • • • •

•

VIRIDANS STREPTOCOCCI Streptococcus mitis, S. salivarius, S. sanguis, S. mutans Major throat normal flora Implicated in Sub neonatal Bacterial Endocarditis STREPTOCOCCUS PNEUMONIAE appears as “LANCET or BULLET-SHAPED DIPLOCOCCI” Normal flora of nasopharynx and oropharynx (URT) Regarded as #1 cause of Adult bacterial meningitis Also, an agent of LOBAR PNEUMONIA and OTITIS MEDIA On plated media, it can develop DOME SHAPED COLONIES after 24 hours incubation o Starting point detection is on BAP o But, dome shaped colonies collapse on prolonged incubation (48 hours) à forming “CRATER COLONIES, NAIL HEAD APPEARING COLONIES or colonies with CHECKER APPEARANCE.” MAJOR VIRULENCE FACTOR is CAPSULE

A.k.a. PYRIDOXAL REQUIRING, SATELLITING STREP THIOL REQUIRING STREPTOCOCCI Abiotrophia & Granulicatella o These are S. adjacens and S. defectives o These are viridans Strep needing VITAMIN B6 for GROWTH.

NEISSERIA • • • •

• • •

Non-motile, aerobic, CAPNOPHILIC COLD SENSITIVE NON-HEMOLYTIC Most species are CHO fermenters o Use of Carbohydrate Utilization test for further classification of Neisseria spp. o (+) YELLOW Develops non-pigmented colonies except: N. flava, N. flavescens, N. sublava All are Catalase Test (+) except: N. elongate All are Cytochrome Oxidase Test (+) except: N. elongata

6

N. GONORRHOEAE Never a normal flora, always a pathogen KIDNEY, COFFEE BEAN SHAPED DIPLOCOCCI intracellular within PMN • VIRULENCE FACTOR: PILI – part of a bacterial cell There are TWO TYPES OF PILI 1. Common pili – for adherence/ attachment to host cell 2. Sex pili – for gene conjugation • Superoxol test (+) – Rapid Test for N. gonorrhoeae o Use of 30% Hydrogen Peroxide • ONLY ferments glucose • Can CAUSE: o Gonorrhea o Ophthalmia neonatorum – a gonorrheal form of conjunctivitis acquired by newborns from an infected mother (gonococcal infection) o Perihepatitis or Fits Hugh Curtis Syndrome – cause sterility.

• •

MEDIA USED FOR CULTIVATING NEISSERIA GONORRHOEAE 1.

THAYER MARTIN MEDIA – enriched chocolate agar (heated blood agar); SELECTIVE MEDIA for Neisseria; contains VCN Vancomycin Inhibits gram NEG Colistin Inhibits gram POS Nystatin Prevents FUNGI

2.

MODIFIED THAYER MARTIN MEDIA – same components as Thayer Martin but with additional antibiotic = VCN – T Vancomycin Inhibits gram NEG Colistin Inhibits gram POS Nystatin Prevents FUNGI TRIMETHOPRIM Prevents swarming organisms (Proteus spp.) LACTATE

3.

MARTIN LEWIS MEDIA – contains VCAT Vancomycin Inhibits gram NEG Colistin Inhibits gram POS ANISOMYCIN Prevents FUNGI Trimethoprim Prevents swarming (Proteus spp.) lactate

4.

5.

NEW YORK CITY MEDIA – contains VCAT Vancomycin Inhibits gram NEG Colistin Inhibits gram POS AMPHOTERICIN Prevents FUNGI B Trimethoprim Prevents swarming lactate (Proteus spp.)

• • • • • • •

N. MENINGITIDIS BEAN SHAPED DIPLOCOCCI; sensitive to SPS Virulence factors: CAPSULE Neufeld Quellung Test (+) Ferments Maltose and Glucose Natural habitat is oro and nasopharynx (normal flora) Specimen to detect carrier state: NASOPHARYNGEAL SWAB Can cause o Bacterial meningitis o Meningococcemia o Waterhouse Friderichsen Syndrome – severe form of Meningococcemia à bleeding of Adrenal Glands

OTHER SPECIES: • N. lactamica: Glucose, Maltose, Lactose (+) & ONPG (+) o ONPG – used to detect late lactose fermenter; used in Enterobacteriaceae o POS: YELLOW • N. sicca: Glucose, Maltose, Sucrose (+) Principle Components Indicator

CTA (CYSTINE TRYPTICASE AGAR) Carbohydrate fermentation Serum + sugar + indicator Phenol red

MORAXELLA CATARRHALIS •

organisms

• • • • • • •

organisms

GC-LECT MEDIUM – contains VCA-T plus Lincomycin; can be used for Mycoplasma hominis and Ureaplasma urealyticum Vancomycin Inhibits gram NEG Colistin Inhibits gram POS Amphotericin B Prevents FUNGI Trimethoprim Prevents swarming organisms (Proteus spp.) lactate LINCOMYCIN Prevent gram POS

•

Develops HOCKEY PUCK COLONIES – thse are colonies remaining intact when pushed across the plate using ino...