LAB 14 – ANTIMICROBIAL SUSCEPTIBILITY TESTING Flashcards
Explain the E-test and why it is used.
- an antibiotic gradient strip
- gives precise and accurate MIC; quantifies antimicrobial susceptibility
- combines dilution and diffusion tests
- but includes anaerobes and fastidious bacteria
MIC - minimum inhibitory concentration
Disk diffusion: Inoculation size ?
10^8 CFU/mL
- compared to 0.5 McFarland standard
Disk diffusion: Ingredients of McFarland standard
- BaCl (barium chloride) + H2SO4 (sulfuric acid) = BaSO4 (barium sulfate)
Disk diffusion: Type of organisms
- Staphylococcus sp.
- Pseudomonas aeruginosa
- Enterobacteriaceae
- NOT ANAEROBES, NOT CO2 BACTERIA
Disk diffusion: Choice of media
Media: Mueller-Hinton agar
- recommended depth of 4mm
Disk diffusion: media constituents/ ingredients
- thymine/ thymidine (folate pathway end-products; antagonistic to trimethoprim and sulfonamides
- para-aminobenzoic acid (PABA); structural analog and antagonist of sulfonamides
- Divalent cations antagonize aminoglycosides, polymyxins, and chelate tetracyclines
- Increased [phosphate] reduces aminoglycoside activity
Disk diffusion: pH
- alkaline pH enhances aminoglycosides, macrolides, and lincomycins
- acidic pH enhances other antibiotics
Disk diffusion: Incubation requirements
Time: 16-18 hours
Temp: at 35°C; MRSA not detectable at higher temp
Atmosphere: in O2; since 5-10% CO2 lowers pH; anaerobic conditions prevent aminoglycoside uptake
Disk diffusion: disk spacing and storage
Spacing: no more than 4 disks
Storage: at -20°C with desiccant to keep disks dry; opened packs can be kept in the fridge
What is meant by modified Kirby Bauer? When would you modify it?
- Modified Kirby Bauer is a reference method used to test the susceptibility of a bacterial isolate in a clinical laboratory
- Disc tests are modified and standardized for each bacteria due to specific growth requirements and antibiotic use/ resistance
Disk diffusion troubleshooting: Zones overlapping
Too many discs in one plate
Disk diffusion troubleshooting: Very large zones for anaerobes
Do not use disk agar diffusion procedure to test anaerobes!
Disk diffusion troubleshooting: All zones are smaller than expected
- agar too thick
- inoculum is too thick
Disk diffusion troubleshooting: “fuzzy” edges of the zone of inhibition
- expired antibiotic disk
- poor confluent streaking
- excessive moisture during incubation
Disk diffusion troubleshooting: Zone sizes universally too large
- inoculum is too light
- agar too thin
- nutritionally poor medium; only use Mueller-Hinton
What is the mechanism of increased resistance to penicillin by pneumococci?
- mutation in penicillin-binding proteins
- NOT B-lactamase
Which organisms that we worked with get modified disc-agar diffusion testing? What are the modifications for each?
Haemophilus influenzae:
- uses Haemophilus Test Medium (HTM): Mueller-Hinton agar supplemented with bovine haematin, NAD, and yeast extract
- 5-7% CO2 for 16-18 hours at 35°C
Neisseria gonorrhoeae:
- GC agar with 1% Isovitalex
- 5-7% CO2 for 20-24 hours at 35°C
Streptococcus pneumoniae:
- Mueller-Hinton agar with 5% defibrinated sheep blood
- 5-7% CO2 for 16-18 hours at 35°C
How would you test antimicrobial susceptibility for an anaerobe?
Do an E test
Define MIC . How is it determined?
MIC = minimum inhibitory concentration
- the lowest concentration of antibiotic that inhibits visible growth using a dilution method
- Mueller-Hinton broth (1.0 mL) with antibiotic + organism (10^8 CFU/L = standard) is serially diluted
- last clear tube = MIC
Define MBC. How is it determined?
MBC: minimum bactericidal concentration
- the smallest concentration of antibiotic which fails to grow a subculture
- clear tubes from MIC dilution are subcultured on a plate
- last plate without growth = MBC
Name 2 antagonists to antibiotic activity in culture media. Explain why they are
antagonists.
- Thymine/ Thymidine:
- end-products of folate pathway that antagonize trimethoprim and sulfonamides - Para-aminobenzoic acid (PABA):
- structural analog to and antagonist of sulfonamides
- antagonists bc they hinder antibiotic activity
Standard Kirby-Bauer Pros vs Cons
Pros:
- flexible antibiotic use
- simple technique
Cons:
- only qualitative; not quantitative
- no fastidious (slow-growing) organisms or anaerobes
- inaccurate in slightly elevated levels of resistance
Dilution method Pros vs Cons
Pros:
- Quantitative; accurate MIC determination
Cons:
- Requires more technique
- Time-consuming
- Higher degree of inherent error
Describe the principle for a test used to detect B-lactamase resistance.
- Nitrocefin is a chromogenic cephalosporin
- When the amide bond of a B-lactam is cleaved by a B-lactamase, the solution changes from yellow to red
- Effective for Staphylococci, Bacteroides sp.
Define inducible and constitutive B-lactamases. Give an example for each.
Inducible= small quantities are present but not detectable
- detectable quantities are produced after continuous exposure to B-lactam (ie. Staphylococcus sp.)
Constitutive = B-lactamase is produced continuously despite presence/ absence of B-lactam (ie. H. influenzae, N. gonorrhoeae)
How does the atmosphere used in susceptibility testing affect the test?
- Some bacteria (S. pneumoniae) require CO2 for initial isolation
- CO2 may reduce pH of Mueller-Hinton agar and alter antibiotic activity
