LAB 14 – ANTIMICROBIAL SUSCEPTIBILITY TESTING

Question 1

Explain the E-test and why it is used.

Answer 1

• 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

Question 2

Disk diffusion: Inoculation size ?

Answer 2

10^8 CFU/mL
- compared to 0.5 McFarland standard

Question 3

Disk diffusion: Ingredients of McFarland standard

Answer 3

• BaCl (barium chloride) + H2SO4 (sulfuric acid) = BaSO4 (barium sulfate)

Question 4

Disk diffusion: Type of organisms

Answer 4

• Staphylococcus sp.
• Pseudomonas aeruginosa
• Enterobacteriaceae
• NOT ANAEROBES, NOT CO2 BACTERIA

Question 5

Disk diffusion: Choice of media

Answer 5

Media: Mueller-Hinton agar
- recommended depth of 4mm

Question 6

Disk diffusion: media constituents/ ingredients

Answer 6

• 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

Question 7

Disk diffusion: pH

Answer 7

• alkaline pH enhances aminoglycosides, macrolides, and lincomycins
• acidic pH enhances other antibiotics

Question 8

Disk diffusion: Incubation requirements

Answer 8

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

Question 9

Disk diffusion: disk spacing and storage

Answer 9

Spacing: no more than 4 disks
Storage: at -20°C with desiccant to keep disks dry; opened packs can be kept in the fridge

Question 10

What is meant by modified Kirby Bauer? When would you modify it?

Answer 10

• 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

Question 11

Disk diffusion troubleshooting: Zones overlapping

Answer 11

Too many discs in one plate

Question 12

Disk diffusion troubleshooting: Very large zones for anaerobes

Answer 12

Do not use disk agar diffusion procedure to test anaerobes!

Question 13

Disk diffusion troubleshooting: All zones are smaller than expected

Answer 13

• agar too thick
• inoculum is too thick

Question 14

Disk diffusion troubleshooting: “fuzzy” edges of the zone of inhibition

Answer 14

• expired antibiotic disk
• poor confluent streaking
• excessive moisture during incubation

Question 15

Disk diffusion troubleshooting: Zone sizes universally too large

Answer 15

• inoculum is too light
• agar too thin
• nutritionally poor medium; only use Mueller-Hinton

Question 16

What is the mechanism of increased resistance to penicillin by pneumococci?

Answer 16

• mutation in penicillin-binding proteins
• NOT B-lactamase

Question 17

Which organisms that we worked with get modified disc-agar diffusion testing? What are the modifications for each?

Answer 17

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

Question 18

How would you test antimicrobial susceptibility for an anaerobe?

Answer 18

Do an E test

Question 19

Define MIC . How is it determined?

Answer 19

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

Question 20

Define MBC. How is it determined?

Answer 20

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

Question 21

Name 2 antagonists to antibiotic activity in culture media. Explain why they are
antagonists.

Answer 21

1. Thymine/ Thymidine:
   - end-products of folate pathway that antagonize trimethoprim and sulfonamides
2. Para-aminobenzoic acid (PABA):
   - structural analog to and antagonist of sulfonamides

• antagonists bc they hinder antibiotic activity

Question 22

Standard Kirby-Bauer Pros vs Cons

Answer 22

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

Question 23

Dilution method Pros vs Cons

Answer 23

Pros:
- Quantitative; accurate MIC determination
Cons:
- Requires more technique
- Time-consuming
- Higher degree of inherent error

Question 24

Describe the principle for a test used to detect B-lactamase resistance.

Answer 24

• 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.

Question 25

Define inducible and constitutive B-lactamases. Give an example for each.

Answer 25

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)

Question 26

How does the atmosphere used in susceptibility testing affect the test?

Answer 26

• Some bacteria (S. pneumoniae) require CO2 for initial isolation
• CO2 may reduce pH of Mueller-Hinton agar and alter antibiotic activity