ANTIBIOGRAMAS COMPLETO

Antibiogram Procedure Overview

Introduction to Antibiograms

• The antibiogram allows healthcare professionals to identify which antibiotics a specific bacterium is sensitive or resistant to, aiding in effective treatment.

Sample Preparation

• Three to five colonies are taken using a swab and introduced into saline solution; some may use enrichment media. Excess liquid is removed by squeezing the swab before reading absorbance at 625 nm.

• If the initial reading exceeds the desired range (0.08 - 0.1), additional saline is added for dilution until the correct value is achieved.

Inoculum Adjustment

• The process of adjusting bacterial concentration continues until readings fall within the target range, ensuring accurate inoculum preparation.

• Once an appropriate inoculum is obtained, it’s mixed well and then spread onto agar plates in three different directions to ensure even distribution.

Agar Plate Preparation

Importance of Agar Height

• The agar medium must be exactly 4 mm thick; too high can lead to false resistance due to poor antibiotic diffusion, while too low can cause false sensitivity.

Plating Technique

• Two agar plates are typically prepared: one for testing resistance mechanisms and another for alternative antibiotic options. Each plate should have bacteria evenly distributed without gaps.

Antibiotic Disk Placement

Disk Application Process

• Antibiotic disks are placed on the agar surface with specific spacing: 1.5 cm from the edge and 2.5 - 3 cm between disks, with a maximum of six disks per standard-sized plate.

Considerations for Different Microorganisms

• The type of microorganism (cocci vs bacilli) influences disk selection and placement strategy during testing.

Final Steps Before Incubation

Securing Disks on Agar Plates

• After placing all disks, gentle pressure ensures they adhere properly before incubating overnight at optimal conditions.

Inoculum Preparation Techniques

Alternative Methods Using Bacteriological Loop

• An alternative method involves using a bacteriological loop instead of a swab for inoculum preparation, which may reduce contamination risks from swab fibers.

Calibration and Reading Absorbance

How to Prepare an Antibiotic Sensitivity Test

Preparation of Inoculum

• The process begins with acclimatizing to the turbidity levels, aiming for a reading between 0.08 and 0.094 before proceeding with inoculation.

• A swab is used for inoculation, ensuring uniformity in height (4 mm) across all media to avoid false resistances and sensitivities.

• Inoculation is performed in three different directions to ensure thorough coverage without leaving gaps that could complicate readings.

Application of Antibiotics

• After inoculating, the swab is discarded, and antibiotics are placed using tweezers while applying slight pressure to prevent contamination.

• Care must be taken not to disturb the antibiotic discs once placed; if they fall or are moved, they should be discarded as this affects concentration diffusion.

Understanding Antibiotic Resistance Mechanisms

• Various antibiotics are introduced including amoxicillin-clavulanic acid and meropenem; understanding their abbreviations helps in identifying resistance mechanisms.

• The importance of noting which antibiotics are sensitive or resistant is emphasized for effective patient treatment.

Reading Results from Antibiogram

• After incubation, results show clear zones around antibiotic discs indicating sensitivity; sizes help classify them as sensitive, intermediate, or resistant.

• Specific attention is given to mechanisms like beta-lactamase production by placing certain discs in a specific order during testing.

Identifying Resistance Patterns

• The "egg effect" indicates resistance when there’s deformation between antibiotic discs due to bacterial activity against penicillins and cephalosporins.

• If resistance patterns indicate failure against third-generation cephalosporins, it suggests careful monitoring of patient response may be necessary.

Antibiotic Resistance Testing and Interpretation

Overview of Antibiotic Testing Procedures

• The speaker discusses the ability to place multiple antibiotics in a single box for simultaneous resistance mechanism testing, emphasizing the importance of reading the antibiogram after 24 hours.

• A method is described where six antibiotics are placed in one box, while another box contains specific antibiotics aimed at identifying resistance mechanisms.

Measurement Techniques for Antibiotic Disks

• Proper measurement techniques are highlighted, including using a ruler or a caliper to measure from edge to edge through the center of the antibiotic disk for accurate readings.

• Reference is made to CLSI guidelines and modifications from previous years, indicating that different bacteria require specific disks for testing sensitivity or resistance.

Interpreting Results for Enterobacteriaceae

• The focus shifts to Enterobacteriaceae, with an example provided using cefoxitin. The speaker explains how to determine if bacteria are sensitive, intermediate, or resistant based on disk diffusion results.

• Specific attention is given to interpreting results from Kirby-Bauer methods versus other methods like SIM (Sulfide Indole Motility), clarifying which data applies in this context.

Classification of Antibiotic Sensitivity

• The classification process involves determining whether the measured value indicates sensitivity (≥18 mm), intermediate, or resistance based on established criteria.

• An example illustrates how results should be documented: sensitive antibiotics are noted alongside their respective measurements for clarity when reporting to medical professionals.

Documenting Resistance Mechanisms

• The final steps involve creating a table categorizing each antibiotic as sensitive, intermediate, or resistant. If any resistance mechanisms are identified (e.g., ESBL production), they should be noted next to the bacterial name.