Download Veterinary Practice Laboratory Unit 1

Microbiology
Unit 7
Chapter 42
Antimicrobial Sensitivity Testing
Copyright © 2015 by Mosby, an imprint of Elsevier Inc. All rights reserved.
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.
2
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
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3
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
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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• Outdated disks should not be used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
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5
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.
6
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
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7
Indirect versus Direct
• Indirect testing is more precise than direct
testing, but results are not available as
quickly
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8
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.
9
Disk Placement
• Dispenser or sterile forceps
•
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
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10
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.
11
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
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12
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.
13
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
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14
Limitations of the Test
• The FDA method is designed for rapidgrowing 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.
15
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
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16
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.
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