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.

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.

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.

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.

Antimicrobial Disks Kept in the refrigerator when not used Outdated disks should not be used Potency of disks monitored with control organisms A disk dispenser for placement obtained from the manufacturer of the disks Caliper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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.