Day 3 (3): Ocular Microbiology Flashcards
Normal flora of the outer eye (conjunctiva and lid margin).
- Staphylococcus epidermidis
- Staphylococcus aureus
- Corynebacterium
- Micrococcus
- Propionibacterium acnes
Most common infectious cause of blepharitis?
- Staphylococcus
- Moraxella
Most common infectious cause of conjunctivitis?
- Staphylococcus aureus
- Streptococcus
- Chlamydia trachomatis
- Neisseria gonorrhoeae
- Haemophilus influenzae
- Moraxella
Most common infectious cause of keratitis?
- Staphylococcus (aureus and epidermidis)
- Streptococcus pneumoniae
- Pseudomonas aeruginosa
- Moraxella
- Fungal (Fusarium, Aspergillus, Candida albicans)
Most common infectious cause of canaliculitis?
Actinomycetes
Hordeolum vs Chalazion
Hordeolum:
- location: at or near an eyelash follicle
- cause: ACUTE BACTERIAL INFECTION of the glands of Zeis or Moll (EXTERNAL/Stye) or Meibomian Gland (INTERNAL)
- presentation: acute redness, swelling and tenderness; pimple-like
- treatment: warm compress, antibiotics
Chalazion:
- location: above or extending beyond eyelashes on upper lid
- cause: CHRONIC, STERILE INFLAMMATION due to a blocked Meibomian or Zeis gland
- may develop from an INTERNAL hordeolum
- presentation: chronic, firm, nontender nodule; larger
- treatment: warm compress, surgery
Mnemonics:
No! Stay back! It HURTS - HORDEOLUM
Dude! Take a CHILL pill - CHALAZION
Bacterial classification according to GRAM STAIN and SHAPE
Coccus = sphere
Bacillus = rod
Gram (+) Cocci:
1. Staphylococcus
2. Streptococcus
3. Enterococcus
Gram (+) Bacilli:
1. Bacillus: (+) spores
2. Corynebacterium: (-) spores
Gram (+) Pleomorphic:
1. Propionibacterium
Gram (+) Filaments:
1. Actinomyces israelli
2. Nocardia asteroides
Gram (-) Cocci:
1. Neisseria
Gram (-) Bacillus:
1. Moraxella
2. Haemophilus
3. Pseudomonas
4. Enterobacter
Gram (-) Spirochetes (spiral, comma):
1. Treponema pallidum
2. Borrelia burgdorferi
Gram (-) Pleomorphic
1. Chlamydia
OTHERS:
AFB: Mycobacteria
Rationale behind Gram staining
Gram Stain
- separates bacteria based on CELL WALL COMPOSITION
Gram POSITIVE (PURPLE)
- 90% peptidoglycan which acts like a mesh that traps crystal violet in between layers
Gram NEGATIVE (RED)
- 10% peptidoglycan BUT high lipid content
What are the steps of Gram Staining?
- Primary Stain: Crystal Violet
- Mordant/Fixant: Iodine
- binds to crystal violet and traps and fixes it in the cell wall - Decolorizing Agent: Acetone/Ethanol
- Gram (+): THICK peptidoglycan layer traps the dye
- Gram (-): outer lipid layer of cell wall is partially dissolved thus crystal violet is washed out from the THIN peptidoglycan layer - Counterstain: Safranin
Culture media classification by function.
- Basic: simple media that supports growth of most NON-FASTIDIOUS bacteria
- okay for most bacteria but cannot isolate - Enriched: extra nutrients added for growth of FASTIDIOUS bacteria (choosy)
- for bacteria with special needs - Selective/Differential: inhibitory agents added to suppress growth of some microorganisms while allowing some to thrive
- antibiotics, dyes, chemicals, pH alteration
- for bacterial isolation
Most basic culture medium
Nutrient Agar
- (+) Growth: NON-FASTIDIOUS bacteria –> mixed colonies
- Contains needs of most bacteria to grow BUT LACKS nutrients needed by FASTIDIOUS bacteria
- FASTIDIOUS bacteria DOES NOT grow:
1. Neisseria
2. Moraxella
3. H. influenzae
4. Streptococcus
What is Blood agar for?
Blood Agar
- Enriched: Nutrient agar (basic) + 5-10% Sheep’s blood
- Differential: (+) Growth and differentiation based on hemolytic activity by HEMOLYSINS
1. Staphylococcus
2. Streptococcus - Hemolysins: lipids and proteins that cause lysis of RBCs by disrupting the cell membrane
- Selective: (-) Growth because of inhibitors in fresh blood
1. Neisseria
2. Haemophilus
Classification of bacteria based on hemolysis of RBCs on blood agar.
- BETA-Hemolytic: CLEAR zone with clear edge around colony = COMPLETE hemolysis
Mnemonic: CLEAR Py (Pie) BAG
- Streptococcus PYogenes (Group A Streptococcus/GAS)
- Beta
- Staphylococcus Aureus
- Streptococcus aGalactiae (Group B Streptococcus/GBS)
- ALPHA-Hemolytic: GREEN (Biliverdin) cloudy zone around = PARTIAL hemolysis
Mnemonic: GREEN VAN
- Viridans streptococcus group
- Alpha
- Streptococcus pNeumoniae
GAMMA-Hemolytic: NO COLOR CHANGE (still red) around =
NO HEMOLYSIS
- Staphylococcus epidermidis and saprophyticus
What is Chocolate agar?
Chocolate Agar
- Blood agar heated to 80 degrees Celsius to:
1. Inactivate inhibitors in fresh blood
2. Release growth factors - Heating causes lysis of RBCs
- (+) Growth: Haemophilus influenzae Type B
What is Thayer-Martin VPN agar?
Thayer-Martin VPN Agar
- Chocolate agar + antibiotics
1. Vancomycin: (-) Gram positive
2. Polymyxin/Colistin: (-) Gram negative
3. Nystatin: (-) fungi - (+) Growth: Neisseria gonorrhoeae and meningitidis
Modified VPN Agar
- (+) Trimethoprim: (-) Proteus
What is the MacConkey and EMB agar?
Similarities:
1. BOTH selective and differential media (only different additives)
2. BOTH allow growth of Gram (-) bacteria ONLY
3. BOTH utilize lactose and dyes to differentiate Gram (-) bacteria
4. BOTH work by the same principle: strength of lactose fermentation to lactic acid causing an increase pH
MacConkey Agar
- Inhibitors: Bile Salts + Crystal Violet –> inhibit Gram (+)
- pH Indicator: Neutral Red –> differentiate lactose vs non-lactose fermenters
- Weak - Strong fermenters: pink to dark red
- Non-fermenters: colorless
EMB Agar
- Inhibitors: Methylene Blue –> inhibit Gram (+)
- pH Indicator: Eosin Y –> differentiate lactose vs non-lactose fermenters
- Weak - Strong fermenters: brown-pink to deep purple to black
- Non-fermenters: colorless
Fermenters: Enterobacteriaceae, E. coli (with green metallic sheen)
Non-fermenters: P. aeruginosa, Salmonella, Shigella
What is the Thioglycolate Broth?
Thioglycolate Broth
- Differentiates bacteria based on oxygen requirements
- Enriched: Sodium thioglycolate –> absorbs oxygen –> anaerobic environment
- Differential: Resazurin –> dye that turns pink if (+) oxygen
- Agar: prevent diffusion of oxygen to lower layers
- Obligate aerobes: NEED oxygen
- topmost layer - Microaerophiles: anaerobes that can tolerate oxygen IN MODERATION
- upper part but not at top because too much oxygen is poison - Facultative anaerobe: aerobes that PREFER OXYGEN but can grow with no oxygen
- mostly at top but can be found everywhere - Aerotolerant: can tolerate any condition thus NO PREFERENCE
- everywhere - Obligate anaerobes: HATE oxygen
- bottommost layer
What is Brain Heart Infusion agar?
BHI Agar
- Enhanced: (+) boiled bovine/porcine heart and brain
- Non-selective
- Growth of FASTIDIOUS organisms
1. Streptococcus
2. Meningococcus/Neisseria meningitides
3. Fungi
What are the other culture media used and organisms that grow on them?
- Potassium Tellurite Agar: Corynebacterium
- Lowenstein-Jansen Agar: Mycobacterium
- Non-Nutrient Agar with E. coli overlay: Acanthamoeba
- alternative to corneal biopsy in keratitis - Saboraud Agar: Fungi
Common stains used.
- Gram Stain
- Gram (+): purple/blue
- Gram (-): red/pink - Giemsa/Wright-Giemsa Stain
- Nucleic acid stain: phosphate group of DNA
- Human cells: purple/blue
- Bacterial cells: red/pink - Acid Fast (Ziehl-Neelsen/Kinyoun) Stains
- Mycobacteria: red/pink
- Others: blue (ZN) or green (K) - Potassium Hydroxide (KOH) Stain
- Fungi (Dermatophytes, Candida) - Calcofluor-White Stain
- Acanthamoeba keratitis - Gomori-Methenamine Silver Stain
- Fungi (BOTH dead and alive)
- Cell wall: brown to black
- Background: green - Periodic Acid Schiff
- Fungi (ALIVE only)
- Cell wall: magenta
- Background: blue
When presented with a Gram (+) cocci, what test is used to determine if its Staphylococcus or Streptococcus?
Catalase Test: catalase breaks down peroxide into oxygen and water
- (+) if with bubbles or oxygen production
Positive: STAPHYLOcoccus (+ grape-like cluster)
Negative: STREPTOcoccus (+ pairs/chains/strips)
How to differentiate Staphylococcus species?
- Coagulase Test: coagulase activates prothrombin and causes fibrin in plasma to clot
- (+) if with clumps = clotting
Positive: S. aureus
Negative: Others
- Novobiocin Test: test for antibiotic resistance
Sensitive/(+) Zone of Inhibition: S. epidermidis
Resistant/(-) Inhibition: S. saprophyticus
How to differentiate Streptococcus species?
Growth pattern on BLOOD AGAR
- Beta-hemolytic: complete hemolysis = clear zone
–> Bacitracin Test
Sensitive/(+) Zone of Inhibition: S. pyogenes (Group A)
Resistant/(-) Zone: S. agalactiae (Group B)
Mnemonic: Clear Py (pie) BAG
PYogenes - Beta - Aureus - aGalactiae
- Alpha-hemolytic: partial hemolysis = green zone (biliverdin)
–> Optochin Test
Sensitive/(+) Zone of Inhibition: S. pneumoniae
Resistant/(-) Zone: Viridans group
Mnemonic: Green VAN
Viridans - Alpha - pNeumoniae
- Gamma-hemolytic: no hemolysis = no change
Antibiotic resistance tests to determine Staphylococcus or Streptococcus species
Novobiocin: Staphylococcus (epidermis vs saprophyticus)
Bacitracin: Beta-hemolytic Streptococcus (pyogenes vs agalactiae)
Optochin: Alpha-hemolytic Streptococcus (pneumoniae vs viridans)
Describe Staphylococcus spp.
Mnemonic: CLUSTER of people posing for a group photo with a CAT wearing GOLD (aureus) medals
Gram POSITIVE cocci - purple/blue
In grape-like CLUSTERS
Facultative anaerobe: aerobe with faculty to become anaerobe
CATalase POSITIVE: (+) bubble formation
- Common infectious agent in EXTERNAL eye infections (especially BLEPHARITIS)
Treatment:
Penicillins, Cephalosporins, Clindamycin, Tobramycin, Erythromycin, Vancomycin, Fluoroquinolones (Levo, Gati, Moxi)
Describe Staphylococcus aureus.
Gram POSITIVE cocci - purple/blue
Grape-like CLUSTERS
Facultative anaerobe
Catalase POSITIVE: (+) bubble formation
Coagulase POSITIVE: (+) clumping or clots
BETA-hemolytic: (+) GOLD pigments
Virulence factors:
1. Coagulase: activates prothrombin and causes fibrin to clot
- fibrin formation around the bacteria protective from phagocytosis
- Hemolysin: destroys RBCs, WBCs, platelets
- tested with blood agar - Leukocidin: destroys WBC
- Panton-Valentine-Leukocidin: in CA-MRSA, (+) recurrent abscess - Penicillinase: destroys B-lactam ring on penicillins
- Exfoliatin: causes skin to slough off (Scalded Skin Syndrome)
- Toxic Shock Syndrome Toxin (TSST-1): cytokine storm
- Enterotoxin: gastroenteritis
- Staphylokinase: thrombolytic to break up blood clots and spread from the initial site of infection
Describe Staphylococcus epidermidis.
Gram POSITIVE cocci - purple/blue
Grape-like CLUSTERS
Facultative anaerobe
Catalase POSITIVE: (+) bubble formation
Coagulase NEGATIVE: (-) clumping
GAMMA-hemolytic
Novobiocin SENSITIVE: (+) zone of inhibition
- Part of normal skin flora
- Problematic in immunocompromised and nosocomial
- (+) Biofilm formation in catheters and prosthetic devices
- Multiple antibiotic resistance
Describe Staphylococcus saprophyticus.
Gram POSITIVE cocci - purple/blue
Grape-like CLUSTERS
Facultative anaerobe
Catalase POSITIVE: (+) bubble formation
Coagulase NEGATIVE: (-) clumping
GAMMA-hemolytic
Novobiocin RESISTANT: (-) zone of inhibition
- UTI in females
Describe Streptococcus spp.
Mnemonic: STRIPtococcus
Gram POSITIVE cocci - purple/blue
In PAIRS/CHAINS/STRIPS
Facultative anaerobe: aerobe with faculty to become anaerobe
CATalase NEGATIVE: (-) bubble formation
Classification based on:
- Hemolytic activity in blood agar
Beta = CLEAR zone = complete hemolysis = pyogenes, agalactiae
Alpha = GREEN zone = partial hemolysis = viridans, pneumoniae
Gamma = NO zone = non-hemolytic
- Lancefield antigen: antigenicity of C carbohydrate on cell wall
Group A: Pyogenes
Group B: Agalactiae
No antigen: Viridans and Pneumoniae
- Common cause of CONJUNCTIVITIS and KERATITIS
Treatment:
Penicillins, Cephalosporins, Clindamycin, Erythromycin, Vancomycin, Fluoroquinolones (Levo, Gati, Moxi)
Describe Streptococcus pyogenes.
Lancefield antigen: Group A
Gram POSITIVE cocci - purple/blue
In CHAINS/STRIPS
Facultative anaerobe/Microaerophile
CATalase NEGATIVE: (-) bubble formation
BETA-hemolytic
Bacitracin SENSITIVE: (+) zone of inhibition
Virulence factors:
- Pyogenes = Pus-producing = microaerophile = skin infections
- Streptolysin O: similar to hemolysin of S. aureus
- destroys RBCs, WBCs, and platelets
- test: Anti-Streptolysin O antibodies - Erythrogenic Toxin: similar to TSST-1 of S. aureus
- cytokine storm - Streptokinase: thrombolytic to break up blood clots and spread from the initial site of infection
- M protein: adherence, antigenicity and anti-phagocytic
Similarities in virulence factors of S. pyogenes and S. aureus
Thrombolytic: Staphylokinase :: Streptokinase
Cytokine Storm: Toxic Shock Syndrome Toxin - 1 :: Erythrogenic Toxin
Hemolysis: Hemolysin :: Streptolysin O
Describe Streptococcus agalactiae.
Lancefield antigen: Group B
Gram POSITIVE cocci - purple/blue
In CHAINS/STRIPS
Facultative anaerobe
CATalase NEGATIVE: (-) bubble formation
BETA-hemolytic
Bacitracin RESISTANT: (-) zone of inhibition
Mnemonic: B for baby (found in vagina)
Describe Streptococcus pneumoniae/Pneumococcus.
Lancefield antigen: NONE
Gram POSITIVE - purple/blue
In PAIRS (diplococci), LANCET-shaped with tapered ends and surrounding CLEAR area
Facultative anaerobe
CATalase NEGATIVE: (-) bubble formation
ALPHA-hemolytic
Optochin SENSITIVE: (+) zone of inhibition
(+) Quellung Reaction: swelling when mixed with antibodies and methylene blue
Mnemonic: P for parents (pneumonia and meningitis in adults)
- vaccinate with Pneumococcal vaccines (PCV13 and PPSV23)
Describe Viridans streptococcus group.
Lancefield antigen: NONE
Gram POSITIVE cocci - purple/blue
In CHAINS/STRIPS
Facultative anaerobe
CATalase NEGATIVE: (-) bubble formation
ALPHA-hemolytic
Optochin RESISTANT: (-) zone of inhibition
Mnemonic: V as in verde/green = alpha-hemolytic
Found in GI tract, gums, teeth, tongue, saliva
- dental infections, endocarditis