14.6 Testing the Effectiveness of Antimicrobials
3 min read•june 18, 2024
Antimicrobial susceptibility testing is crucial for determining the effectiveness of antibiotics against specific bacteria. Methods like Kirby-Bauer disk diffusion and / tests help identify which drugs will work best for treating infections.
Understanding these techniques is vital for healthcare professionals. They guide treatment decisions, help combat , and ensure patients receive the most appropriate and effective antimicrobial therapy for their specific infections.
Antimicrobial Susceptibility Testing
Kirby-Bauer disk diffusion test
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- Standardized bacterial inoculum evenly spread on an agar plate
- Paper disks impregnated with specific concentrations of antibiotics placed on the agar surface
- During incubation, antibiotic diffuses from the disk into the surrounding agar creates a concentration gradient
- Susceptible bacteria unable to grow in the presence of the antibiotic resulting in a clear around the disk
- Diameter of the measured in millimeters
- Measured zone of inhibition compared to standardized interpretive charts categorizes bacteria as susceptible, intermediate, or resistant to the antibiotic
- Larger zones of inhibition indicate greater susceptibility ()
- Smaller or no zones of inhibition suggest resistance (methicillin-resistant Staphylococcus aureus)
- Interpretation of results based on guidelines set by the
MIC vs MBC in antimicrobial testing
- lowest concentration of an antimicrobial agent prevents visible growth of a microorganism after overnight incubation
- Determined by observing the lowest concentration of the antimicrobial results in no visible bacterial growth
- Values used to determine susceptibility of bacteria to antimicrobials and guide treatment decisions ()
- lowest concentration of an antimicrobial agent required to kill 99.9% of the initial bacterial inoculum
- Determined by subculturing from the tests that show no visible growth and assessing colony formation
- Values indicate the concentration necessary to achieve a effect ()
- MIC and MBC values may differ for a given antimicrobial and bacterial strain
- Antimicrobial may inhibit growth at a lower concentration (MIC) than is required to kill the bacteria (MBC)
- Ratio of MBC to MIC provides insight into whether an antimicrobial is or ( vs )
Methods of antimicrobial susceptibility determination
- , , and methods used to determine MIC values for antimicrobials
- Macrobroth dilution (broth dilution)
- Prepare two-fold serial dilutions of the antimicrobial in broth medium using test tubes
- Add standardized bacterial inoculum to each tube
- Incubate tubes overnight
- Lowest concentration of the antimicrobial prevents visible bacterial growth is the MIC
- Labor-intensive and requires larger volumes of reagents compared to other methods
- Microdilution
- Utilize 96-well microtiter plates to prepare serial dilutions of the antimicrobial
- Add standardized bacterial inoculum to each well
- Incubate plate overnight
- Determine MIC by observing the lowest concentration that inhibits visible bacterial growth
- More efficient and economical than macrobroth dilution allows for testing of multiple antimicrobials simultaneously
- Etest ()
- Place plastic strip impregnated with a predefined gradient of antimicrobial concentrations on an agar plate inoculated with a standardized bacterial suspension
- During incubation, antimicrobial diffuses into the agar creates a concentration gradient
- Read MIC where the edge of the elliptical zone of inhibition intersects the Etest strip
- Convenient method for determining MICs particularly for fastidious or slow-growing bacteria (Streptococcus pneumoniae)
-
- Prepare agar plates containing different concentrations of antimicrobials
- Inoculate standardized bacterial suspension onto the plates
- Incubate and observe for bacterial growth
- MIC is the lowest concentration that inhibits visible growth
- Choice of method depends on factors such as the number of antimicrobials and bacterial strains to be tested, available resources, and the level of automation desired
- Microdilution and Etest more commonly used in clinical settings due to their efficiency and ease of use
- Macrobroth dilution often used in research settings or for confirmatory testing
Antibiotic Resistance
- Occurs when bacteria develop mechanisms to survive in the presence of antibiotics
- Can be intrinsic (naturally occurring) or acquired through genetic mutations or horizontal gene transfer
- Contributes to treatment failures and the emergence of multidrug-resistant organisms
- Necessitates ongoing surveillance and development of new antimicrobial agents
Key Terms to Review (42)
Agar Dilution: Agar dilution is a method used to determine the minimum inhibitory concentration (MIC) of antimicrobial agents against a specific microorganism. It involves incorporating various concentrations of an antimicrobial agent into agar media and then inoculating the agar plates with a standardized bacterial suspension to assess the effectiveness of the antimicrobial.
Antibiogram: An antibiogram is a laboratory test result that shows the susceptibility of bacterial strains to various antimicrobial agents. It helps guide the selection of appropriate antibiotics for treatment.
Antibiotic Resistance: Antibiotic resistance is the ability of bacteria and other microorganisms to withstand the effects of antibiotics, rendering these drugs ineffective in treating infections. This phenomenon is a growing global health concern that has significant implications across various aspects of microbiology, including prokaryote habitats, antimicrobial discovery and chemotherapy, and the treatment of bacterial infections.
Antimicrobial Gradient Method: The antimicrobial gradient method is a technique used to test the effectiveness of antimicrobial agents against microorganisms. It involves creating a concentration gradient of the antimicrobial agent and observing the response of the target microorganisms to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).
Antimicrobial susceptibility testing (AST): Antimicrobial susceptibility testing (AST) determines the effectiveness of specific antimicrobial agents against bacteria or fungi. It helps guide appropriate treatment by identifying resistant and susceptible strains.
Bactericidal: Bactericidal agents are antimicrobial drugs that kill bacteria. They differ from bacteriostatic agents, which inhibit bacterial growth without directly killing the bacteria.
Bactericidal: Bactericidal refers to the ability of a substance or agent to kill bacteria. It is a crucial concept in the field of antimicrobial chemotherapy, the mechanisms of antibacterial drugs, and the testing of antimicrobial effectiveness.
Bacteriostatic: Bacteriostatic agents inhibit the growth and reproduction of bacteria without killing them. They rely on the immune system to eliminate the inhibited bacteria.
Bacteriostatic: Bacteriostatic refers to the ability of a substance or agent to inhibit the growth and reproduction of bacteria without necessarily killing them. This term is particularly relevant in the context of antimicrobial chemotherapy, the mechanisms of antibacterial drugs, and the testing of antimicrobial effectiveness.
Broth Dilution: Broth dilution is a method used to determine the minimum inhibitory concentration (MIC) of an antimicrobial agent against a specific microorganism. It involves serially diluting the antimicrobial agent in a liquid growth medium, known as broth, and then inoculating the dilutions with a standardized microbial culture to assess the antimicrobial's effectiveness.
Carbapenemase: Carbapenemase is an enzyme produced by certain bacteria that hydrolyzes carbapenem antibiotics, rendering them ineffective. This mechanism contributes significantly to antibiotic resistance.
Chloramphenicol: Chloramphenicol is a broad-spectrum antibiotic that inhibits bacterial protein synthesis, making it an effective treatment against a wide range of bacterial infections. It is particularly relevant in the contexts of modern cell theory and the testing of antimicrobial effectiveness.
Ciprofloxacin: Ciprofloxacin is a broad-spectrum antibiotic belonging to the fluoroquinolone class, effective against various bacterial infections. It operates by inhibiting DNA gyrase and topoisomerase IV, enzymes crucial for bacterial DNA replication.
Ciprofloxacin: Ciprofloxacin is a broad-spectrum fluoroquinolone antibiotic used to treat a variety of bacterial infections. It works by inhibiting the DNA gyrase and topoisomerase IV enzymes, which are essential for bacterial DNA replication and transcription, ultimately leading to cell death. Ciprofloxacin has been widely used in the treatment of infections since its development and approval in the 1980s.
Clinical and Laboratory Standards Institute (CLSI): The Clinical and Laboratory Standards Institute (CLSI) is a non-profit organization that develops and publishes voluntary consensus standards and guidelines for the healthcare community. It plays a crucial role in the context of testing the effectiveness of antimicrobials by providing standardized protocols and recommendations for antimicrobial susceptibility testing.
Colistin: Colistin is a polymyxin antibiotic used as a last-resort treatment for multidrug-resistant Gram-negative bacterial infections. It disrupts the bacterial cell membrane, leading to cell death.
Etest: The Etest is a quantitative antimicrobial susceptibility testing method that provides a simple and accurate way to determine the minimum inhibitory concentration (MIC) of antimicrobial agents against a specific microorganism. It is commonly used in the context of testing the effectiveness of antimicrobials, as described in section 14.6 of the chapter.
Gentamicin: Gentamicin is an aminoglycoside antibiotic used to treat serious bacterial infections by inhibiting protein synthesis. It is effective against a range of Gram-negative and some Gram-positive bacteria.
Gentamicin: Gentamicin is an aminoglycoside antibiotic used to treat various bacterial infections. It is particularly effective against gram-negative bacteria and plays a crucial role in the mechanisms of antibacterial drugs and the testing of antimicrobial effectiveness.
Kirby-Bauer disk diffusion test: The Kirby-Bauer disk diffusion test is a method used to determine the sensitivity of bacteria to specific antimicrobial agents. It involves placing antibiotic-impregnated disks on an agar plate inoculated with the test organism and measuring zones of inhibition.
Kirby-Bauer Disk Diffusion Test: The Kirby-Bauer disk diffusion test is a standardized method used to determine the susceptibility of bacteria to various antimicrobial agents. It is a widely employed technique in clinical microbiology laboratories to guide the selection of appropriate antibiotics for the treatment of bacterial infections.
Klebsiella pneumoniae: Klebsiella pneumoniae is a Gram-negative, encapsulated bacterium that can cause severe infections, particularly in the lungs and urinary tract. It is known for its resistance to multiple antibiotics, making infections difficult to treat.
Macrobroth Dilution: Macrobroth dilution is a quantitative antimicrobial susceptibility testing method used to determine the minimum inhibitory concentration (MIC) of an antimicrobial agent against a specific microorganism. It involves preparing a series of dilutions of the antimicrobial agent in a liquid growth medium and inoculating each dilution with a standardized bacterial suspension.
MBC: MBC, or Minimum Bactericidal Concentration, is the lowest concentration of an antimicrobial agent that is required to kill a specific microorganism, rather than just inhibiting its growth. It is an important measure in testing the effectiveness of antimicrobials and determining appropriate treatment strategies.
Methicillin-resistant Staphylococcus aureus (MRSA): Methicillin-resistant Staphylococcus aureus (MRSA) is a type of Staphylococcus bacteria that has developed resistance to methicillin and other beta-lactam antibiotics. It is known for causing difficult-to-treat infections in humans.
Methicillin-Resistant Staphylococcus aureus (MRSA): Methicillin-Resistant Staphylococcus aureus (MRSA) is a strain of the common bacterium Staphylococcus aureus that has developed resistance to many antibiotics, including the antibiotic methicillin. This resistance makes MRSA infections particularly challenging to treat and a significant public health concern.
The term MRSA is directly relevant to the topics of 14.5 Drug Resistance, 14.6 Testing the Effectiveness of Antimicrobials, and 16.3 Modes of Disease Transmission. MRSA's resistance to antibiotics is a prime example of drug resistance, while the ability to effectively test for and identify MRSA is crucial for controlling its spread and transmission.
MIC: MIC, or Minimum Inhibitory Concentration, is a key metric used in the context of testing the effectiveness of antimicrobials. It represents the lowest concentration of an antimicrobial agent that is able to inhibit the visible growth of a microorganism in vitro.
Microdilution: Microdilution is a laboratory technique used to determine the minimum inhibitory concentration (MIC) of antimicrobial agents against specific microorganisms. It involves serial dilutions of an antimicrobial agent in a small volume of growth medium to assess the lowest concentration that can inhibit the growth of a tested microorganism.
Minimal Bactericidal Concentration: The minimal bactericidal concentration (MBC) is the lowest concentration of an antimicrobial agent that is required to kill a specific microorganism, rather than just inhibiting its growth. It is an important metric for evaluating the effectiveness of antimicrobial agents in the context of testing their ability to eliminate bacterial populations.
Minimal bactericidal concentration (MBC): Minimal bactericidal concentration (MBC) is the lowest concentration of an antimicrobial agent required to kill a particular bacterium. It is determined after identifying the minimum inhibitory concentration (MIC).
Minimal inhibitory concentration (MIC): Minimal Inhibitory Concentration (MIC) is the lowest concentration of an antimicrobial agent that inhibits visible growth of a microorganism after overnight incubation. It helps determine the effectiveness of antibiotics against specific pathogens.
Minimum Inhibitory Concentration: The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial agent that is required to inhibit the visible growth of a microorganism under defined in vitro conditions. It is a crucial parameter in understanding the effectiveness of antimicrobial agents and their potential for controlling microbial growth in various contexts, such as infection treatment, disinfection, and assessing drug resistance.
Penicillin: Penicillin is a class of antibiotics derived from the Penicillium fungi that revolutionized the treatment of bacterial infections. It works by inhibiting the synthesis of the bacterial cell wall, making it a crucial discovery in the history of chemotherapy and antimicrobial development.
Polymyxins: Polymyxins are a class of antibiotics that target the cell membranes of Gram-negative bacteria. They are especially effective against multi-drug resistant strains.
Spectrophotometer: A spectrophotometer is an analytical instrument used to measure the amount of light that a sample absorbs. It quantifies concentrations of compounds in solutions by measuring absorbance at specific wavelengths.
Tetracycline: Tetracycline is a broad-spectrum antibiotic used to treat a wide variety of bacterial infections. It works by inhibiting protein synthesis in bacteria, preventing them from growing and multiplying.
Therapeutic dosage: Therapeutic dosage is the quantity of a drug required to achieve the desired therapeutic effect without causing significant side effects. It is determined through clinical trials and testing for efficacy and safety.
Tigecycline: Tigecycline is a broad-spectrum glycylcycline antibiotic derived from minocycline. It is used to treat various bacterial infections, particularly those resistant to other antibiotics.
Vancomycin: Vancomycin is a glycopeptide antibiotic used to treat serious bacterial infections caused by Gram-positive bacteria. It inhibits cell wall synthesis by binding to the D-alanyl-D-alanine terminus of cell wall precursor units.
Vancomycin: Vancomycin is a glycopeptide antibiotic used to treat severe bacterial infections, particularly those caused by Gram-positive bacteria that are resistant to other antibiotics. It is an important tool in the fight against antimicrobial resistance and is a key consideration across various topics in microbiology and infectious disease management.
Zone of inhibition: The zone of inhibition is the clear region around an antimicrobial agent on an agar plate where bacterial growth has been prevented. It is used to measure the efficacy of the antimicrobial substance.
Zone of Inhibition: The zone of inhibition refers to the clear area around an antimicrobial agent or disinfectant on a growth medium where microbial growth is prevented or inhibited. It is a key indicator used to assess the effectiveness of these agents in controlling or eliminating microbial populations.