5. Pruebas de susceptibilidad a los antimicrobianos Método de dilución en caldo

Introduction to Antimicrobial Susceptibility Testing

Importance of Antimicrobial Susceptibility Testing (AST)

• Understanding the appropriate treatment for diseases caused by microorganisms is critical for achieving therapeutic success.

• AST allows monitoring the behavior of microorganisms against various antimicrobials over time, providing insights into their effectiveness.

• Techniques such as broth dilution and agar dilution are essential for assessing antimicrobial activity without requiring sophisticated equipment.

Broth Dilution Method Overview

• The broth dilution method is a standard approach used in AST, involving bacterial cultures with increasing concentrations of antibiotics to measure in vitro activity against specific bacterial isolates.

• There are two main types: broth dilution and agar dilution; within broth dilution, there are microdilution and macrodilution variants. The results help determine the Minimum Inhibitory Concentration (MIC).

Serial Dilutions Explained

• For testing antivirals, double serial dilutions are employed, starting from a higher concentration and decreasing gradually (e.g., 64 µg/mL down to 0.5 µg/mL).

• Agar dilution involves preparing plates with known concentrations of antimicrobials that decrease gradually across each plate. In contrast, broth dilutions differ mainly in volume: macro (2 mL) vs micro (100 µL).

Reference Organizations for AST Methods

• International organizations like EUCAST and CLSI provide reference methods, quality control parameters, and interpretation criteria for antimicrobial susceptibility tests.

• In Latin America, CLSI recommendations are followed for human health applications, while EUCAST guidelines are adhered to in Europe. Examples from CLSI documents will be utilized throughout this module.

Workflow of Broth Dilution Method

Steps in Performing Broth Dilution Tests

• The CLSI M07 document outlines four main steps: preparation of antibiotics and serial dilutions, inoculum preparation, incubation, and result reading. Each step is crucial for accurate testing outcomes.

Preparation of Antibiotic Solutions

• Start by preparing stock solutions of antibiotics at a concentration ten times greater than what will be tested (e.g., 1,280 µg/mL), considering solubility and potency requirements based on the antibiotic type being tested.

Conducting Serial Dilutions

Microbial Inoculum Preparation and Antimicrobial Testing

Microbial Inoculum Adjustment

• The microbial inoculum is adjusted to 0.5 on the Macfarland scale using a nephelometer or spectrophotometer. This ensures accurate bacterial load for testing.

• The isolate used must be pure, fresh, and no older than 24 hours prior to testing to maintain reliability in results.

Microdilution Methodology

• For microdilution, after adjusting the inoculum, a dilution of 1:20 is performed, resulting in a bacterial load of 5x10^6 CFU/mL (colony-forming units per milliliter). Subsequently, 10 microliters of diluted inoculum are added to each well, achieving a final concentration of 5x10^4 CFU/well.

Macrodilution Process

• In the macrodilution method, the adjusted inoculum at 0.5 on the Macfarland scale is diluted 1:150 to achieve a microbial load of 1x10^6 CFU/mL before adding it to tubes containing antibiotic dilutions and control growth tubes.

• A further dilution of 1:2 will yield a final concentration of 5x10^5 CFU/mL in test tubes while maintaining sterility controls without bacterial inoculation. Incubation occurs at approximately 35°C for 16-20 hours.

Reading Results and Interpretation

• After incubation, visual readings determine the minimum inhibitory concentration (MIC), defined as the lowest concentration that visibly inhibits microorganism growth. Proper interpretation relies on established cut-off points from international guidelines like EUCAST and CLSI for susceptibility or resistance assessment.

• Epidemiological cut-off points differ from clinical ones as they are based solely on in vitro data without proven clinical relevance by regulatory bodies like CLSI. They lack information regarding pharmacokinetics and pharmacodynamics related to MIC distributions.

Quality Control Measures

• Quality control is critical throughout all steps; extra wells or tubes verify inoculum growth and sterility controls while reference strains appropriate for tested antibiotics are utilized for accuracy checks against contamination risks due to poor laboratory practices. Contaminated broth necessitates discarding entire assays due to potential inaccuracies in results caused by improper solvent use with antibiotics that may not dissolve adequately in water based on their physicochemical properties.

Conclusion on Antimicrobial Testing Techniques

• The broth dilution technique allows determination of MIC through incubation across various dilutions within enrichment broth media containing antimicrobial agents, providing quantitative insights into effective concentrations against microorganisms over time within clinical settings which aids better selection of antimicrobials while monitoring resistance patterns effectively over time.

Future Considerations

• Identifying genotypes associated with resistance mechanisms becomes crucial when encountering resistant strains; understanding techniques available for such identification can enhance treatment strategies significantly moving forward.