1. Chapter 42 Antimicrobial Sensitivity Testing
Microbiology Unit 7
Chapter 42
Antimicrobial Sensitivity Testing
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

2. Antimicrobial Sensitivity
Performed to determine the susceptibility or resistance to specific antimicrobial drugs
Helps veterinarian choose the most appropriate antimicrobial for the patient
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

3. Agar Diffusion Method Most commonly preferred method
Uses paper disks impregnated with antimicrobials
Quantitative and requires the measurement of inhibitory zone sizes
Gives an estimate of susceptibility
Concentrations on disks correlate with therapeutic levels of the drugs in tested animals
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

4. Agar Diffusion Method (cont.)
Modified Kirby-Bauer technique
U.S. Food and Drug Administration method
International collaborative study
Minimum inhibitory concentration (MIC)
The smallest concentration that can inhibit growth
Disks with varying concentrations
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

5. Antimicrobial Disks Kept in the refrigerator when not used
Outdated disks should not be used
Potency of disks monitored with control organisms
A disk dispenser for placement obtained from the manufacturer of the disks
Caliper for measuring
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

6. Antimicrobial Disks (cont.)
Inoculation of a thioglycollate or trypticase soy broth tube at the time the plates are streaked is recommended
In case the in-house culture is inconclusive
Broth cultures can be sent to a reference lab for confirmation
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

7. Indirect Sensitivity Testing
Requires that colony samples be taken from a culture plate and subcultured in broth media
Incubate to achieve turbidity to match a standardized 0.5 McFarland suspension
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

8. Indirect versus Direct
Indirect testing is more precise than direct testing, but results are not available as quickly
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

9. Direct Testing Method
Application of undiluted samples (urine) directly to a Mueller-Hinton plate
Not as precise
Reasonable results if only one organism is present
Interpret with caution if multiple organisms are present
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

10. Disk Placement Dispenser or sterile forceps 10 to 15 mm apart
Flamed and cooled between each use
10 to 15 mm apart
Avoid overlapping of ZOI
Tamp disk into the agar with a sterile swab
Incubate, inverted and aerobic at 37º C, within 15 minutes of disk placement
Stacks of four or fewer
If taller, the middle plates take longer to get to temperature
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

11. Zone of Inhibition
Measurement is compared to a chart of ZOI to determine the relative resistance of the bacterium to the antimicrobial
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

12. Reading of ZOI Plates read after a constant time period
Usually 18 to 24 hours
Prolonged incubation may alter the size of ZOI
Zones read from the bottom of the plate using a caliper, transparent ruler, or template
Measured to the nearest millimeter
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

13. Interpretation of Zone Sizes
Zone sizes are divided into two major categories
Resistant
Susceptible
Implies the organism is susceptible to ordinary doses of antimicrobials
Intermediate susceptibility
Implies the organism is susceptible to ordinary doses of antimicrobials when the drug is concentrated in the urine or tissues
Or the drug may be used for treatment of systemic infections if a high dose is safe
Size not indicative of efficacy
Some drugs do not diffuse through agar
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

14. Control Organisms Susceptible reference organisms
S. aureus (ATCC 25923)
E. coli (ATCC 25922)
Should be tested regularly in parallel with each batch of antimicrobial susceptibility tests
Used to check growth-supporting capability of medium, potency of disks, and other factors
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

15. Limitations of the Test
The FDA method is designed for rapid-growing bacteria
Caution needed for slower-growing bacteria
Strains of staphylococci are resistant to methicillin and other similar drugs
Routine tests cannot be relied upon to detect these strains
A reduced zone diameter or no zone is evidence of methicillin resistance
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

16. Colony Count
The presence of pathogenic bacteria does not necessarily indicate infection
Colony counts on cultured urine can help support a diagnosis of UTI
After incubation all colonies are counted and multiplied by 100 to determine the number of colony-forming units per millimeter of urine
Guideline for significance
>1000 CFUs in cystocentesis sample
>10,000 CFUs in catheter sample
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.

17. Summary
Antibiotic sensitivity testing is performed to determine the resistance or susceptibility of bacteria to specific antimicrobials
Kirby-Bauer agar diffusion method most commonly used
Application of undiluted samples directly to a Hinton-Mueller plate is direct sensitivity testing
Indirect sensitivity testing requires a colony taken from a culture plate
ZOI is measured from the underside of the plate
The presence of bacteria does not necessarily indicate infection
A colony count on a cultured urine sample can help support a diagnosis of UTI
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.