Unit 1: Antibiotic susceptibility testing
Applied Bacteriology notes · PTU syllabus (BMLS307-18)
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Unit summary
Antibiotic susceptibility testing tells doctors which drug will work. This unit covers antibiotics, media for testing, inoculum preparation and standardisation, control strains, choice of antibiotics, MIC and MBC, the Kirby–Bauer and Stokes methods, and tests for β-lactamase production.
After this unit you can
- Define antibiotics and classify them
- Prepare and standardise an inoculum and use control strains
- Perform Kirby–Bauer and Stokes disc diffusion tests
- Determine MIC and MBC and detect β-lactamases
PTU syllabus topics
- Definition of antibiotics
- culture media for AST
- inoculum preparation and standardization
- control bacterial strains
- choice of antibiotics
- MIC and MBC concepts and determination
- Kirby-Bauer and Stokes methods
- tests for beta-lactamase production
- 1
Prepare inoculum
Match 0.5 McFarland standard
- 2
Swab Mueller-Hinton agar evenly
- 3
Place antibiotic discs
- 4
Incubate at 35–37 °C for 16–18 hours
- 5
Measure zones of inhibition
In mm
- 6
Interpret
Sensitive, intermediate or resistant (CLSI or EUCAST)
Topic 1
Antibiotics
- Antibiotic: a substance produced by a microorganism (or made synthetically — antimicrobial) that kills or inhibits other microorganisms. Bactericidal drugs kill (penicillins, aminoglycosides); bacteriostatic drugs inhibit growth (tetracyclines, macrolides).
Cell wall synthesis
Penicillins, cephalosporins, carbapenems, vancomycin
Peptidoglycan
Protein synthesis
Aminoglycosides, tetracyclines, macrolides, linezolid
30S or 50S ribosome
Nucleic acid synthesis
Fluoroquinolones, rifampicin
DNA gyrase; RNA polymerase
Folate synthesis
Sulphonamides, trimethoprim
Metabolic pathway
Cell membrane
Polymyxins (colistin)
Outer membrane
Topic 2
Media, inoculum and control strains
- Medium
- Mueller–Hinton agar, 4 mm deep, pH 7.2–7.4; with 5% blood for fastidious organisms
- Inoculum
- 3–5 colonies in saline or broth adjusted to 0.5 McFarland standard (about 1.5 × 10⁸ CFU/mL)
- Inoculation
- Within 15 minutes; swab in three directions for a confluent lawn
- Discs
- Applied within 15 minutes, 24 mm apart; stored at −20 °C or 2–8 °C
- Incubation
- 35 ± 2 °C for 16–18 hours
- S. aureus ATCC 25923
- Gram-positive disc tests
- E. coli ATCC 25922
- Enterobacteriaceae
- P. aeruginosa ATCC 27853
- Non-fermenters
- E. faecalis ATCC 29212
- Enterococci, MIC
- McFarland 0.5 standard: 0.05 mL of 1.175% barium chloride in 9.95 mL of 1% sulphuric acid.
Topic 3
Choice of antibiotics
- Follow CLSI or EUCAST guidelines and the hospital antibiogram: first-line drugs tested routinely, second-line on request or if resistance is found; test drugs relevant to the site (nitrofurantoin only for urine) and organism.
Topic 4
Kirby–Bauer and Stokes methods
Comparison
Zone diameter compared with CLSI interpretive tables
Zone compared with a control strain on the same plate
Layout
Whole plate with test lawn
Test and control strains inoculated side by side
Result
Sensitive, intermediate or resistant from breakpoints
Sensitive if the test zone is equal to or within 3 mm of the control
Use
Standard worldwide
Simpler where standardisation is difficult
- Principle: the antibiotic diffuses from the disc creating a concentration gradient; the zone of inhibition is inversely related to the MIC.
Topic 5
MIC and MBC
- MIC (minimum inhibitory concentration)
- Lowest concentration with no visible growth after overnight incubation
- MBC (minimum bactericidal concentration)
- Lowest concentration killing 99.9% of the inoculum (subculture of clear tubes)
Broth macrodilution
Doubling dilutions in tubes
Reference; laborious
Broth microdilution
96-well plates
Standard for colistin; automated systems
Agar dilution
Drug in agar plates
Many strains at once
E-test (gradient strip)
Strip with a gradient; read where the ellipse meets the strip
Easy; read MIC directly
Example
Tubes with 64, 32, 16, 8, 4, 2, 1 µg/mL: growth at 2 and 1, clear from 4 upwards → MIC = 4 µg/mL. Subculture: growth from 4, none from 8 → MBC = 8 µg/mL.
Topic 6
Tests for β-lactamase production
Chromogenic cephalosporin (nitrocefin)
Hydrolysis changes the colour
Yellow to red within minutes
Iodometric
Penicilloic acid reduces iodine
Starch–iodine blue colour disappears
Acidimetric
Penicilloic acid lowers pH
Phenol red turns yellow
ESBL phenotypic confirmation
Cefotaxime or ceftazidime alone vs with clavulanic acid
Zone increase of 5 mm or more with clavulanate
Carbapenemase detection
Modified carbapenem inactivation method (mCIM), Carba NP
Loss of meropenem activity
- MRSA: detected with the cefoxitin disc (zone 21 mm or less resistant) or PCR for mecA.
Key terms
- Antibiotic susceptibility testing
- Testing which drugs inhibit an isolate
- McFarland standard
- Turbidity standard for inoculum density
- MIC
- Lowest drug concentration inhibiting visible growth
- MBC
- Lowest concentration killing 99.9% of bacteria
- ESBL
- Extended-spectrum β-lactamase hydrolysing cephalosporins
Quick revision
- Antibiotic classes and targets; bactericidal vs bacteriostatic.
- Mueller–Hinton agar; 0.5 McFarland; ATCC control strains.
- CLSI or EUCAST; antibiogram.
- Kirby–Bauer vs Stokes; zone vs MIC.
- MIC methods; MBC; nitrocefin, iodometric, acidimetric; ESBL, carbapenemase, MRSA tests.
Important exam questions
Practice questions written to the PTU exam pattern for this unit's syllabus: short answers (Section A style) and long answers (Sections B and C style).
Short-answer questions
- Q1.Why is Mueller–Hinton agar used?
- Q2.What does 0.5 McFarland correspond to?
- Q3.Name two control strains.
- Q4.Distinguish MIC and MBC.
- Q5.How is ESBL confirmed?
- Q6.Which disc detects MRSA?
Long-answer questions
- Q1.Describe the Kirby–Bauer disc diffusion method.
- Q2.Explain MIC and MBC determination.
- Q3.Describe tests for β-lactamase production.
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