Antimicrobial Susceptibility Testing Methods

• The resource focuses on methods for antimicrobial susceptibility testing (AST).

Microbiological Investigation

• The process involves pathogenic microorganism identification, such as Escherichia coli and Staphylococcus aureus.
• Antimicrobial susceptibility testing (AST), also known as antibiogram, is performed.
• Antibiotic therapy is guided by the results of AST.

Resistance to Antimicrobial Agents

• Intrinsic resistance is a fundamental property of a microorganism, predictable and present in most strains long before antibiotic exposure, with Gram-negative bacteria and vancomycin as one example.
• Acquired resistance is unpredictable, arises from spontaneous chromosomal mutations or the acquisition of extrachromosomal elements like plasmids and transposons, explaining the need for susceptibility testing.

Standards for Antimicrobial Susceptibility Testing

• The Clinical and Laboratory Standards Institute (CLSI) was a global organization and was known as NCCLS until 2005.
• EUCAST was formed in 1997, is organized by ESCMID, ECDC, and national breakpoint committees.
• EUCAST's Development Laboratory for antibacterial agents resides in Sweden, while the antifungal agents lab is in Denmark.
• Established on April 1st, 2011, Poland mandates EUCAST compliance.

Key Websites

• www.eucast.org provides free standards
• www.clsi.org distributes payable standards: includes methodology, breakpoints for interpretation, and Quality Control ranges.

Antimicrobial Susceptibility Testing (AST) Goal

• AST seeks to determine if an organism shows acquired resistance to agents that could be used in therapy.
• An antibiogram reflects the pattern of an organism's susceptibility to antimicrobial agents in vitro.

Categories of Susceptibility

• Susceptible (S): A microorganism is categorized as susceptible with standard dosing regimen when a high likelihood of therapeutic success with a standard dosing regimen of the agent is proven.
• Intermediate (I): A microorganism is susceptible with increased exposure when a high likelihood of therapeutic success can be reached only with increased exposure by adjusting the dosing regimen or concentration at the infection site.
• Resistant (R): An organism is categorized as resistant when there is a high likelihood of therapeutic failure, even with increased exposure.
• Exposure relates to administration mode, dose, dosing interval, and infusion time.

Antimicrobial Susceptibility Tests

• Qualitative tests categorize isolates as susceptible, intermediate (susceptible with increased exposure), or resistant.
• Quantitative tests determine the Minimum Inhibitory Concentration (MIC) and also categorize isolates as susceptible, intermediate (susceptible with increased exposure), or resistant.

Qualitative Tests

• Disk diffusion.
• Screening (e.g., chromagar).
• Molecular methods.

Quantitative Tests

• Tests use a strip impregnated with an antimicrobial concentration gradient (e.g., Etest).
• Broth dilution.
• Agar dilution.

Disk Diffusion (Kirby-Bauer)

• Disk diffusion involves paper disks with known antimicrobial concentrations.
• An antimicrobial agent diffuses into the agar, inhibiting susceptible microorganism growth.

Media for Disk Diffusion

• Mueller-Hinton (MH) agar is used for non-fastidious organisms.
• Mueller-Hinton (MH-F) agar + 5% horse blood + 20 mg/l β-NAD is used for fastidious organisms.

Inoculum Preparation

• Inoculum consists of bacterial or fungal cells used to inoculate a new medium.
• Suspend one to several colonies in 0.85% saline solution for even, visible turbidity.
• Well-isolated colonies should also be selected.

Inoculum Standards

• Requires an inoculum suspension equivalent to a 0.5 McFarland standard, approximately 1-2 x 10⁸ CFU/mL for Escherichia coli.
• The McFarland standard measures turbidity in bacterial suspensions.

Turbidity Measurement

• Turbidity can be measured by the eye using turbidity standards.
• Turbidity can be measured by a photometric device - densitometer (preferably).
• Adjust the turbidity by adding more bacteria or saline solution.

Inoculation of Plates

• The inoculum must spread evenly over the entire surface.
• This is achieved by inoculating in three directions (two 60-degree turns).

Antimicrobial Disk Application

• Disks must be applied within 15 minutes of inoculation.
• Application method by forceps or by dispenser are often used.

Disk Spacing

• Disks should be spaced to prevent overlapping inhibition zones.
• The minimum center-center distance is 24 mm.
• Disks should be no closer than 10 - 15 mm from the edge of the petri dish.
• Use a maximum of six disks on a 90 mm petri dish or 12 disks on a 150 mm plate.

Disk Contact

• Disks must have firm and even contact with the medium surface.
• Press lightly with forceps and also avoid moving disks post-contact to not disrupt the instantaneous diffusion.

The 15-15-15 Minute Rule

• Use the inoculum within 15 minutes of preparation (never beyond 60 minutes).
• Apply disks within 15 minutes of inoculating plates.
• Start incubation of application of disks within 15 minutes of application of disks.

After Incubation

• After incubation, confluent growth is often achieved
• Following the development of inhibition zones (no growth) surrounding the disks.

Reading Zones

• Zone diameters are measured via ruler, calliper, or automated zone reader.
• Zone edges are read at the point of complete inhibition as judged by the naked eye.

Plate Reading

• MH plates get read reading them from the back against a black background illuminated with reflected light.
• MH-F plates are read from the front with the lid removed and illuminated with reflected light.

Interpreting Zones

• Measured zone diameters are interpreted into susceptibility categories (S, I, R).
• This categorization is done via tables containing breakpoints (by EUCAST).
• Breakpoints defines inhibition zone sizes and MIC values in quantitative methods categorizing isolates as S, I, or R.

Screening Methods

• Screening methods aids to identify specific microbial resistance phenotypes.
• A solid/liquid medium contains a defined concentration of the selected antibiotic.
• A material is inoculated onto/into the medium, in which microorganisms with a specific phenotype of resistance is sought and incubated.
• Interpretation of the process involves determination of growth/lack of growth of microorganisms.

Screening Method Action

• Growth usually occurs in the form of colonies of a specific color when a chromogen soluble colourless molecule is added to the medium.
• When the chromogen is added to the medium (due to enzymatic activity of the microorganism), colored colonies form .
• Manufacturer's instructions and standard methods of determining the drug susceptibility of microorganisms should be followed when reading screening samples.

Examples of Screening

• Enterococcus faecalis (blue colonies), Enterococcus faecium (purple colonies) identification on chromogenic medium is for the detection of VRE/GRE.
• Escherichia coli (pale pink colonies)
• Klebsiella pneumoniae (celadon colonies) on chromogenic medium aids with the process of CRE detection.

Detection of Resistance Genes

• The detection of resistance genes include mecA and mecC genes in staphylococci, van genes in enterococci, bla, vim, imp genes in Enterobacterales rods, and erm genes in Gram-positive cocci.
• Methods to identify resistance genes includes PCR (single or multiplex), the use of real-time PCR, the use of LAMP (loop mediated isohermal amplification) and hybridization.

Etest

• Antimicrobial agent is applied in gradient to a test strip that gets applied to medium.
• Plate is inoculated with microorganism as in disk diffusion and agent diffuses away from strip to inhibit growth, after incubation, an inhibition ellipse is typically observed
• The zone edge intersects the strip at the MIC value, and this aids with identification.

Broth Dilution Testing

• Various concentrations of agent in broth are tested (typically done with doubling dilutions).
• The MIC (lowest concentration required to visibly inhibit growth) gets defined.
• Incubation time can take as little as 18±2h.

Differences in Dilutions

• Macrodilution testing volume is >1.0 ml
• Microdilution testing volume is 0.05-0.1 ml.

Automated Systems and Their Action

• Automated systems require manual preparation of isolate suspension.
• Systems also need to be completely automated for inoculation, interpretation, and data managment.
• VITEK 2 Compact (bioMérieux) and Phoenix (Becton Dickinson) are two systems
• 6-13 h is usually needed

Agar Dilutions

• Varying concentrations of tested substance are incorporated into agar and prepared for the assay.
• Separately, inoculum of tested microbe is standardized and spotted onto the agar plates.
• After incubation time, MIC is analyzed to determine the lowest tested isolate that will visibly inhibit growth:
• This experiment if typically performed as experiment for research purposed.

Multidrug Resistance (MDR)

• MDR is non-susceptibility to at least one agent in three or more antimicrobial categories (groups)

Extensively Drug-Resistance (XDR)

• XDR is non-susceptibility to at least one agent in all but two or fewer antimicrobial categories where bacterial isolates remain susceptible to only one or two categories.

Pandrug-Resistance (PDR)

• PDR is non-susceptibility to all agents in all antimicrobial categories and often presents as an superbug.

Explore antimicrobial susceptibility testing (AST) methods for identifying pathogenic microorganisms like E. coli and S. aureus. Learn about intrinsic and acquired resistance to antimicrobial agents. Understand the importance of AST in guiding antibiotic therapy and the role of organizations like CLSI in standardizing testing.