Unit 10: Antibiotic sensitivity of microorganism

I. Orientation

Antibiotic sensitivity testing, also called antimicrobial susceptibility testing, determines whether a microorganism is inhibited by a particular antimicrobial agent under standardized laboratory conditions. The result supports selection of effective treatment and helps detect resistance. The central principle is that a measured response, such as an inhibition zone or minimum inhibitory concentration, must be compared with validated interpretive standards for the organism, drug, dose, and testing method.

• Antibiotic sensitivity: The degree to which a microorganism is inhibited or killed by an antimicrobial agent.
• Antimicrobial resistance: The ability of a microorganism to survive or grow despite an antimicrobial concentration that would normally inhibit susceptible organisms.
• Standardization: Inoculum density, medium composition, agar depth, incubation, disk potency, and reading criteria must be controlled.
• Reference standards: Breakpoints are obtained from recognized standards such as CLSI or EUCAST; they are not inferred from zone size alone.
• Clinical categories: Results are commonly reported as susceptible, intermediate or susceptible with increased exposure, and resistant, depending on the standard used.
• Quantitative basis: The minimum inhibitory concentration, or MIC, is the lowest antimicrobial concentration that visibly prevents growth.
• Biological limitation: An in-vitro result predicts activity under test conditions but does not by itself account for drug distribution, host immunity, infection site, or toxicity.

II. Antibiotic sensitivity testing — methods, procedure, and interpretation

Antibiotic sensitivity testing is a comparative laboratory procedure in which a standardized bacterial suspension is exposed to selected antimicrobial agents. The most commonly taught screening method is the Kirby–Bauer disk diffusion technique, while dilution and gradient methods provide MIC values.

A. To study the antibiotic sensitivity pattern of microorganisms

The purpose of this study is to determine the response pattern of an isolated microorganism to several antibiotics by observing growth inhibition around antimicrobial disks or measuring the lowest inhibitory concentration.

• Test organism: Use a pure, freshly isolated culture obtained from a clinically or environmentally relevant specimen.
  • A mixed culture can produce overlapping zones and misleading results.
  • Confirm purity by colony morphology, Gram staining, and appropriate biochemical identification before testing.
• Basic principle: A paper disk containing a defined amount of antibiotic releases the drug into Mueller–Hinton agar.
  • The drug concentration is highest near the disk and decreases with distance.
  • If the organism is inhibited, a clear circular zone develops around the disk.
• Materials: Typical materials include Mueller–Hinton agar, sterile swabs, antibiotic disks, sterile forceps or a disk dispenser, a turbidity standard, incubator, ruler or caliper, and quality-control strains.
• Safety: Treat all unknown isolates as potentially pathogenic.
  • Wear a laboratory coat, gloves, and eye protection.
  • Disinfect the work area and sterilize or discard cultures and contaminated materials according to institutional biosafety procedures.

B. Kirby–Bauer disk diffusion method

The Kirby–Bauer method estimates susceptibility from the diameter of zones of inhibition produced by standardized antibiotic disks on agar.

• Medium: Use Mueller–Hinton agar because its controlled pH, low concentrations of inhibitors, and reproducible diffusion properties support standardized testing.
  • The usual agar depth is approximately 4 mm; excessively thick agar may reduce zone diameters, whereas thin agar may enlarge them.
  • The surface should be dry but not excessively desiccated.
• Inoculum preparation: Select several similar colonies from an 18–24-hour culture and suspend them in sterile saline or broth.
  • Adjust turbidity to 0.5 McFarland, approximately 1–2 × 10⁸ CFU/mL for many bacteria.
  • A visibly heavy suspension may produce falsely small zones and false resistance.
• Inoculation: Dip a sterile swab into the suspension, remove excess fluid against the tube wall, and streak the entire agar surface in several directions.
  • Rotate the plate approximately 60 degrees between streakings to produce an even bacterial lawn.
  • Finally, swab around the rim to avoid an unseeded margin.
• Disk placement: Apply disks after the surface has absorbed excess moisture, pressing each disk firmly onto the agar.
  • Space disks sufficiently to prevent overlapping zones; the exact number depends on plate diameter and disk spacing.
  • Record the antibiotic name, disk concentration, and plate identification.
• Incubation: Invert the plate and incubate under the conditions specified for the organism and standard.
  • Many routine nonfastidious bacteria are incubated at about 35 ± 2°C for 16–18 hours in ambient air.
  • Fastidious organisms, anaerobes, and unusual species require different media or atmospheric conditions.
• Measurement: Measure the complete diameter of each clear zone in millimetres, including the disk diameter.
  • Read the back of the plate with reflected light when appropriate.
  • Examine for colonies within a zone, swarming, double zones, or an uneven lawn, because these may indicate contamination, mixed culture, or a resistant subpopulation.
• Worked example: If an ampicillin disk produces a 12 mm zone, the result is not labelled susceptible merely because a zone exists. The 12 mm value must be compared with the current breakpoint for that organism and disk potency.

C. Dilution and gradient methods

Dilution methods determine the MIC by exposing the organism to a series of known antibiotic concentrations. They are especially useful when a quantitative value is required.

• Broth microdilution: Prepare wells containing serial twofold concentrations of antibiotic, inoculate each with a standardized suspension, and incubate.
  • The concentration series may be 0.25, 0.5, 1, 2, 4, and 8 µg/mL.
  • The MIC is the lowest well without visible growth.
• Broth macrodilution: The same principle is performed in larger tubes rather than microplate wells.
  • It is conceptually simple but uses more reagent and is less convenient for routine high-volume testing.
• Agar dilution: Incorporate different antibiotic concentrations into agar, spot standardized inocula on the surfaces, and identify the lowest concentration preventing growth.
  • This method can be highly reproducible but is labor-intensive.
• Gradient diffusion: An antibiotic gradient strip is placed on an inoculated agar surface.
  • The MIC is read where the edge of the elliptical inhibition zone intersects the concentration scale.
  • Trailing growth or an indistinct ellipse requires interpretation according to the method’s instructions.
• MIC significance: MIC is a measured concentration, not automatically a treatment dose.
  • Its clinical meaning depends on the organism, infection site, achievable drug exposure, dosing regimen, and breakpoint.

D. Factors affecting the sensitivity pattern

Reliable results depend on controlling variables that alter antimicrobial diffusion or bacterial growth.

• Inoculum size: Too many cells produce smaller zones; too few produce larger zones.
  • Standardize turbidity promptly because bacterial growth changes the suspension over time.
• Agar composition: Excess calcium or magnesium can alter activity of some agents, particularly aminoglycosides against certain organisms.
  • Incorrect pH can change drug stability or bacterial growth.
• Agar depth: A 4 mm depth is a reference condition for disk diffusion; deviations change diffusion distance.
• Disk storage: Moisture, heat, and expired disks can reduce antibiotic potency.
  • Store disks in sealed containers under the manufacturer’s recommended conditions and allow cold disks to reach room temperature before opening to limit condensation.
• Incubation: Temperature, time, atmosphere, and plate orientation affect growth and zone size.
  • Delayed incubation after disk placement can allow early growth and alter diffusion patterns.
• Culture purity: A contaminating organism may create additional colonies or a second growth pattern, making the result invalid.
• Reading error: Measure zones from the nearest whole millimetre and use transmitted or reflected light as required for the organism and drug.

E. Interpretation and reporting

Interpretation converts a laboratory measurement into a standardized category that can guide antimicrobial selection.

• Susceptible: The organism is likely to respond when the recommended regimen produces adequate exposure at the infection site.
• Intermediate: Under CLSI terminology, activity may be uncertain or may occur when drug concentration is unusually high; under EUCAST, “I” means susceptible with increased exposure.
  • The reporting laboratory should identify which standard and terminology it uses.
• Resistant: The organism is unlikely to respond to the agent under standard exposure because the measured result falls in the resistant range.
• Breakpoints: Compare the zone diameter or MIC with the current breakpoint table for the exact organism–drug combination.
  • A breakpoint is not universal: the same 18 mm zone may have different meanings for different organisms or antibiotics.
• Intrinsic resistance: Some organisms naturally lack susceptibility to particular drugs because of cell structure or inherent resistance mechanisms.
  • Such agents may be excluded from testing or reporting.
• Inducible or acquired resistance: Resistance may result from enzymes, altered targets, reduced permeability, or efflux pumps.
  • Examples include beta-lactamase production, altered penicillin-binding proteins, and vancomycin resistance mechanisms.
• Special phenotypic tests: Certain patterns require confirmatory testing, such as tests for extended-spectrum beta-lactamase or carbapenemase production.
  • These tests should be interpreted using validated laboratory protocols rather than zone appearance alone.
• Report format: Record the organism, specimen or isolate identifier, antimicrobial name, disk content or MIC, measured result, interpretive category, method, and standard used.
  • Avoid reporting an antibiotic as active merely because it was included on the plate.

F. Quality control and sources of error

Quality control verifies that the test system performs within accepted limits before patient or research results are released.

• Reference strains: Common control organisms include Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 for disk diffusion, and Pseudomonas aeruginosa ATCC 27853.
  • Their zone diameters or MICs must fall within published acceptable ranges.
• Control failure: If a control result is outside its range, investigate medium, disks, inoculum, incubation, measurement, and culture purity before reporting test isolates.
• Common false resistance: Heavy inoculum, low-potency disks, excessive agar depth, or inadequate incubation can reduce zones.
• Common false susceptibility: Light inoculum, thin agar, excessive antimicrobial concentration, or incorrect reading can enlarge zones.
• Unexpected patterns: A result inconsistent with organism identification should prompt repeat testing and verification rather than immediate clinical reporting.
• Reproducibility: Use the same validated procedure, equipment, and documentation for successive tests so results can be compared over time.

G. Applications and limitations

Sensitivity testing is valuable for therapy guidance, resistance surveillance, infection control, and evaluation of local antibiotic patterns, but it has defined limits.

• Clinical application: Results help clinicians choose an effective agent and avoid unnecessary broad-spectrum therapy.
• Surveillance application: Aggregated patterns reveal increasing resistance within a hospital, community, food system, or environmental population.
• Epidemiological value: Similar unusual resistance profiles may support investigation of transmission or an outbreak.
• Therapeutic limitation: A susceptible result does not guarantee cure if the drug cannot reach the infection site, the patient cannot tolerate it, or source control is inadequate.
• Methodological limitation: Disk diffusion gives a zone diameter, whereas dilution gives an MIC; the two values should not be compared without their appropriate interpretive tables.
• Biological limitation: Biofilms, intracellular organisms, slow-growing bacteria, and mixed infections may behave differently in vivo from a standardized planktonic culture.
• Reporting limitation: Test only clinically relevant antibiotics and report results according to current standards, because inappropriate testing can encourage ineffective or unnecessarily broad treatment.