4. Pruebas de susceptibilidad a los antimicrobianos: Método de difusión de Disco

Introduction to Disk Diffusion Method for Antibiotic Resistance Testing

Overview of Antibiotic Resistance

• Antibiotic resistance in bacteria is a significant public health risk, exacerbated by the intensive use of antimicrobials in food production and human medicine.

• The development of resistance limits the effectiveness of therapeutic treatments for infectious diseases, highlighting the need to monitor bacterial resistance profiles against various antibiotics.

Importance of Testing Methods

• There are seven methods available for testing antibiotic susceptibility, including macro and micro dilution tests; however, this session focuses on the disk diffusion method.

• Developed by Kirti Bauer in 1966, the disk diffusion technique is widely used due to its simplicity and cost-effectiveness despite requiring several days for results. It helps determine bacterial resistance profiles to optimize treatment strategies.

International Guidelines and Standards

Key References

• The primary international references for this technique are from the Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). These organizations provide standardized recommendations essential for accurate testing procedures.

• Laboratories in Latin America typically follow CLSI guidelines while interpreting results, noting slight differences between CLSI and EUCAST criteria regarding resistant or susceptible classifications. Thus, it’s crucial to adhere strictly to one guideline without mixing them.

Methodology of Disk Diffusion Technique

Basic Principles

• The disk diffusion method involves observing zones of inhibition around antibiotic-impregnated disks placed on agar plates inoculated with a standardized concentration of bacteria (approximately 10^8 CFU/mL). This visualizes bacterial growth inhibition due to antibiotics.

Required Materials

• Essential materials include non-selective media like broth and agar for culturing bacteria, saline solution (0.85% NaCl) for standardizing microbial concentrations, and a spectrophotometer or McFarland standard for achieving a 0.5% concentration level during inoculation preparation.

Workflow Stages

General Workflow Steps

1. Preparation of Media: Cultivation media must be prepared adequately before starting the test.

2. Inoculum Preparation: Standardized inocula from both test strains and quality control organisms are prepared.

3. Disk Placement: Impregnated disks are placed onto agar plates followed by incubation.

Preparation and Inoculation of Agar Plates

Media Preparation

• The media should be prepared according to the manufacturer's instructions, with myoliginto agar dispensed in Petri dishes to a depth of approximately 4 mm. This corresponds to about 25 ml for 90 mm dishes, 31 ml for 100 mm dishes, and around 70-71 ml for 150 mm dishes.

Surface Conditions

• It is crucial that the agar surface is dry before inoculation; visible water droplets can compromise results. If moisture is present, Petri dishes should be left uncovered at room temperature overnight or at a higher temperature for a shorter duration.

Inoculum Preparation

• For evaluating new plates, strains must be assessed by culturing on soy agar one day prior to inoculum preparation. Using old strains can lead to inaccurate results.

• The inoculum concentration should reach approximately 10^8 colony-forming units per milliliter. A sterile swab is used to collect fresh colonies from non-selective agar and suspended in saline until homogenous.

Adjusting Density

• The density of the suspension is adjusted using a spectrophotometer to achieve a target of 0.5 McFarland units by adding saline if too dense or more colonies if too dilute.

Inoculation Process

• Both the myoliginto agar and sensitivity discs must be at room temperature before inoculation. Sufficient time should be allowed after removing them from refrigeration.

Inoculating Agar Plates

Application Technique

• An applicator stick (isopuestery) dipped in the standardized inoculum ensures proper application without excess fluid that could lead to over-inoculation.

Rotational Inoculation

• During inoculation, rotate the swab over the agar surface at an angle of approximately 60 degrees in multiple directions (vertical, horizontal, diagonal).

Timing for Disc Placement

• After inoculating the agar, there’s a critical window of about 15 minutes to place sensitivity discs; otherwise, bacterial growth may begin affecting results.

Placement and Incubation Guidelines

Disc Application Importance

• Discs must make perfect contact with the agar surface for effective antibiotic diffusion; movement post-placement can cause variable concentrations around discs.

Number of Discs Used

• Use more than six discs on smaller plates (90 mm), while limiting antibiotics to two when using larger plates (150 mm), as overlapping inhibition zones complicate result interpretation.

Storage Conditions for Discs

• Store discs according to manufacturer guidelines away from light and moisture to maintain potency and ensure accurate inhibition zone measurements.

Incubation Procedures

Incubation Timing

• Incubation should commence no later than 15 minutes after disc placement; this prevents premature antibiotic diffusion which could yield inaccurately large inhibition zones.

Plate Handling During Incubation

• Plates should be inverted during incubation ensuring that discs remain attached firmly without detaching from the agar surface.

Interpreting Results Post-Incubation

Growth Expectations

Quality Control in Antimicrobial Susceptibility Testing

Importance of Inhibition Zone Measurement

• It is crucial to review the inhibition zones of quality control strains and ensure they fall within acceptable ranges as per guidelines from CBCI or EUCAST. The plates are visually inspected at a distance of approximately 30 cm against a dark background.

Measuring Inhibition Diameter

• The diameter of the inhibition zone is measured, considering the area where complete growth inhibition is observed. Results are compared with thresholds that classify organisms as susceptible, intermediate, or resistant according to CBCI or EUCAST standards.

Recommendations for Accurate Results

• To avoid growth within inhibition zones that could affect result readings, it’s essential to conduct result interpretation within established limits. If using CLSA guidelines, the MSN document contains tables with cutoff points for various bacterial species.

Quality Control Strains

• Reliable results require consistent use of quality control strains such as Echerichia coli, Enterococcus faecalis, Staphylococcus aureus, and Pseudomonas aeruginosa. These strains must be kept viable and stored at -70 degrees Celsius in glycerol while being cultured weekly on non-selective media to verify purity.

Ensuring Compliance with Guidelines

• Quality control results should always align with intervals reported by CLSA or EUCAST guidelines. If changing suppliers or acquiring new lots of media used for testing (e.g., Ginton), preliminary tests must confirm that quality control strain inhibition zones remain within specified ranges.

Key Principles in Antimicrobial Sensitivity Testing

Method Overview

• The method's principle involves observing and measuring the inhibition zone generated around antibiotic discs placed on bacterial cultures containing specific antibiotic concentrations. This technique helps identify resistant strains effectively without requiring sophisticated equipment.

Adhering to Timing Rules

• When applying this technique, it's vital to follow the 15-15-15 rule: inoculate no more than 15 minutes after preparing the inoculum; place discs no more than 15 minutes after inoculation; incubate no more than 15 minutes after placing discs. Measurements should also be taken within established timeframes to determine susceptibility accurately based on chosen guidelines (CLSA/EUCAST).

Conclusions on Antibiotic Resistance Testing

Insights into Resistance Profiles

• The disk diffusion method allows for identifying resistant strains against certain antibiotics (e.g., multiple resistances against beta-lactams may indicate beta-lactamase presence). This test can infer genetic resistance profiles but does not specify which genes are involved in observed resistance patterns.

Clinical Implications