What Factors Influence The Size Of The Zone Of Inhibition Produced By A Chemical

Learning Objectives

• Depict how the Kirby-Bauer disk diffusion exam determines the susceptibility of a microbe to an antibacterial drug.
• Explain the significance of the minimal inhibitory concentration and the minimal bactericidal concentration relative to the effectiveness of an antimicrobial drug.

Testing the effectiveness of antimicrobial drugs confronting specific organisms is important in identifying their spectrum of activity and the therapeutic dosage. This blazon of exam, by and large described as antimicrobial susceptibility testing (AST), is unremarkably performed in a clinical laboratory. In this section, we will discuss common methods of testing the effectiveness of antimicrobials.

The Kirby-Bauer Disk Improvidence Examination

The Kirby-Bauer disk diffusion test has long been used as a starting point for determining the susceptibility of specific microbes to various antimicrobial drugs. The Kirby-Bauer assay starts with a Mueller-Hinton agar plate on which a confluent lawn is inoculated with a patient's isolated bacterial pathogen. Filter paper disks impregnated with known amounts of antibacterial drugs to exist tested are then placed on the agar plate. As the bacterial inoculum grows, antibiotic diffuses from the circular disk into the agar and interacts with the growing bacteria. Antibacterial activity is observed as a articulate circular zone of inhibition around the drug-impregnated disk, similar to the deejay-diffusion assay depicted in Figure 1 in Testing the Effectiveness of Antiseptics and Disinfectants. The bore of the zone of inhibition, measured in millimeters and compared to a standardized nautical chart, determines the susceptibility or resistance of the bacterial pathogen to the drug.

There are multiple factors that make up one's mind the size of a zone of inhibition in this analysis, including drug solubility, charge per unit of drug diffusion through agar, the thickness of the agar medium, and the drug concentration impregnated into the disk. Due to a lack of standardization of these factors, interpretation of the Kirby-Bauer deejay diffusion analysis provides only express data on susceptibility and resistance to the drugs tested. The assay cannot distinguish between bacteriostatic and bactericidal activities, and differences in zone sizes cannot be used to compare drug potencies or efficacies. Comparing of zone sizes to a standardized chart volition merely provide information on the antibacterials to which a bacterial pathogen is susceptible or resistant.

Think nearly It

• How does one use the information from a Kirby-Bauer assay to predict the therapeutic effectiveness of an antimicrobial drug in a patient?

Antibiograms: Taking Some of the Guesswork Out of Prescriptions

Unfortunately, infectious diseases don't take a time-out for lab work. Equally a result, physicians rarely have the luxury of conducting susceptibility testing before they write a prescription. Instead, they rely primarily on the empirical evidence (i.east., the signs and symptoms of disease) and their professional feel to brand an educated estimate as to the diagnosis, causative amanuensis(s), and drug virtually probable to exist constructive. This approach allows treatment to begin sooner so the patient does non have to look for lab test results. In many cases, the prescription is constructive; however, in an historic period of increased antimicrobial resistance, it is condign increasingly more hard to select the most appropriate empiric therapy. Selecting an inappropriate empiric therapy not only puts the patient at gamble but may promote greater resistance to the drug prescribed.

Recently, studies have shown that antibiograms are useful tools in the controlling process of selecting appropriate empiric therapy. An antibiogram is a compilation of local antibody susceptibility data broken downwardly by bacterial pathogen. In a November 2014 study published in the journal Infection Control and Infirmary Epidemiology, researchers determined that 85% of the prescriptions ordered in skilled nursing facilities were decided upon empirically, but merely 35% of those prescriptions were deemed advisable when compared with the eventual pathogen identification and susceptibility profile obtained from the clinical laboratory. Notwithstanding, in one nursing facility where use of antibiograms was implemented to direct selection of empiric therapy, appropriateness of empiric therapy increased from 32% earlier antibiogram implementation to 45% later implementation of antibiograms.^[1] Although these information are preliminary, they do advise that health-care facilities can reduce the number of inappropriate prescriptions by using antibiograms to select empiric therapy, thus benefiting patients and minimizing opportunities for antimicrobial resistance to develop.

Dilution Tests

Effigy 1. In a dilution test, the lowest dilution that inhibits turbidity (cloudiness) is the MIC. In this example, the MIC is eight μg/mL. Broth from samples without turbidity tin be inoculated onto plates lacking the antimicrobial drug. The lowest dilution that kills ≥99.9% of the starting inoculum is observed on the plates is the MBC. (credit: modification of piece of work by Suzanne Wakim)

As discussed, the limitations of the Kirby-Bauer disk diffusion test do not allow for a direct comparison of antibacterial potencies to guide selection of the best therapeutic option. All the same, antibacterial dilution tests tin exist used to determine a particular drug'south minimal inhibitory concentration (MIC), the lowest concentration of drug that inhibits visible bacterial growth, and minimal bactericidal concentration (MBC), the lowest drug concentration that kills ≥99.9% of the starting inoculum. Determining these concentrations helps place the correct drug for a particular pathogen. For the macrobroth dilution analysis, a dilution serial of the drug in broth is made in test tubes and the same number of cells of a exam bacterial strain is added to each tube (Effigy 1). The MIC is determined by examining the tubes to discover the lowest drug concentration that inhibits visible growth; this is observed every bit turbidity (cloudiness) in the broth. Tubes with no visible growth are so inoculated onto agar media without antibiotic to make up one's mind the MBC. Generally, serum levels of an antibacterial should exist at least three to v times in a higher place the MIC for handling of an infection.

The MIC assay can also be performed using 96-well microdilution trays, which allow for the use of small volumes and automated dispensing devices, too as the testing of multiple antimicrobials and/or microorganisms in 1 tray (Figure 2). MICs are interpreted as the everyman concentration that inhibits visible growth, the aforementioned as for the macrobroth dilution in exam tubes. Growth may also be interpreted visually or by using a spectrophotometer or similar device to detect turbidity or a color alter if an appropriate biochemical substrate that changes color in the presence of bacterial growth is also included in each well.

Figure 2. A microdilution tray can too be used to determine MICs of multiple antimicrobial drugs in a single assay. In this example, the drug concentrations increase from left to correct and the rows with clindamycin, penicillin, and erythromycin take been indicated to the left of the plate. For penicillin and erythromycin, the lowest concentrations that inhibited visible growth are indicated by cherry-red circles and were 0.06 μg/mL for penicillin and 8 μg/mL for erythromycin. For clindamycin, visible bacterial growth was observed at every concentration upwardly to 32 μg/mL and the MIC is interpreted as >32 μg/mL. (credit: modification of work by Centers for Disease Control and Prevention)

The Etest is an alternative method used to determine MIC, and is a combination of the Kirby-Bauer disk improvidence test and dilution methods. Similar to the Kirby-Bauer assay, a confluent lawn of a bacterial isolate is inoculated onto the surface of an agar plate. Rather than using circular disks impregnated with ane concentration of drug, however, commercially available plastic strips that contain a gradient of an antibacterial are placed on the surface of the inoculated agar plate (Figure 3). As the bacterial inoculum grows, antibiotic diffuses from the plastic strips into the agar and interacts with the bacterial cells. Because the rate of drug diffusion is directly related to concentration, an elliptical zone of inhibition is observed with the Etest drug gradient, rather than a circular zone of inhibition observed with the Kirby-Bauer assay. To interpret the results, the intersection of the elliptical zone with the gradient on the drug-containing strip indicates the MIC. Because multiple strips containing different antimicrobials can be placed on the same plate, the MIC of multiple antimicrobials tin can be determined meantime and directly compared. However, unlike the macrobroth and microbroth dilution methods, the MBC cannot be adamant with the Etest.

Figure 3. The Etest can be used to make up one's mind the MIC of an antibiotic. In this Etest, vancomycin is shown to have a MIC of ane.v μg/mL against Staphylococcus aureus.

Recollect virtually Information technology

• Compare and contrast MIC and MBC.

Clinical Focus: Nakry, Resolution

This case concludes Nakry's story that started in History of Chemotherapy and Antimicrobial Discovery, Mechanisms of Antibacterial Drugs, and Drug Resistance.

Nakry'due south UTI was probable acquired by the catheterizations she had in Vietnam. About bacteria that crusade UTIs are members of the normal gut microbiota, but they can cause infections when introduced to the urinary tract, equally might have occurred when the catheter was inserted. Alternatively, if the catheter itself was not sterile, bacteria on its surface could have been introduced into Nakry'south body. The antimicrobial therapy Nakry received in Kingdom of cambodia may besides accept been a complicating factor because it may have selected for antimicrobial-resistant strains already nowadays in her body. These leaner would have already independent genes for antimicrobial resistance, either acquired past spontaneous mutation or through horizontal cistron transfer, and, therefore, had the all-time evolutionary advantage for adaptation and growth in the presence of the antimicrobial therapy. As a result, one of these resistant strains may have been subsequently introduced into her urinary tract.

Laboratory testing at the CDC confirmed that the strain of Klebsiella pneumoniae from Nakry's urine sample was positive for the presence of NDM, a very active carbapenemase that is showtime to sally every bit a new trouble in antimicrobial resistance. While NDM-positive strains are resistant to a wide range of antimicrobials, they accept shown susceptibility to tigecycline (structurally related to tetracycline) and the polymyxins B and E (colistin).

To forbid her infection from spreading, Nakry was isolated from the other patients in a carve up room. All hospital staff interacting with her were advised to follow strict protocols to preclude surface and equipment contamination. This would include peculiarly stringent mitt hygiene practices and conscientious disinfection of all items coming into contact with her.

Nakry's infection finally responded to tigecycline and somewhen cleared. She was discharged a few weeks after admission, and a follow-upward stool sample showed her stool to be free of NDM-containing Grand. pneumoniae, meaning that she was no longer harboring the highly resistant bacterium.

Key Concepts and Summary

• The Kirby-Bauer disk diffusion test helps decide the susceptibility of a microorganism to various antimicrobial drugs. Nonetheless, the zones of inhibition measured must be correlated to known standards to determine susceptibility and resistance, and do not provide data on bactericidal versus bacteriostatic action, or let for direct comparison of drug potencies.
• Antibiograms are useful for monitoring local trends in antimicrobial resistance/susceptibility and for directing appropriate selection of empiric antibacterial therapy.
• There are several laboratory methods available for determining the minimum inhibitory concentration (MIC) of an antimicrobial drug confronting a specific microbe. The minimal bactericidal concentration (MBC) tin also be determined, typically as a follow-up experiment to MIC determination using the tube dilution method.

Multiple Choice

In the Kirby-Bauer disk diffusion test, the _______ of the zone of inhibition is measured and used for interpretation.

1. bore
2. microbial population
3. circumference
4. depth

Show Reply

Reply a. In the Kirby-Bauer deejay diffusion test, the diameter of the zone of inhibition is measured and used for interpretation.

Which of the following techniques cannot exist used to decide the minimum inhibitory concentration of an antimicrobial drug against a particular microbe?

1. Etest
2. microbroth dilution test
3. Kirby-Bauer disk diffusion test
4. macrobroth dilution test

Show Reply

Answer c. The Kirby-Bauer disk improvidence test cannot be used to determine the minimum inhibitory concentration of an antimicrobial drug against a particular microbe.

The utility of an antibiogram is that it shows antimicrobial susceptibility trends

1. over a large geographic expanse.
2. for an individual patient.
3. in inquiry laboratory strains.
4. in a localized population.

Evidence Respond

Answer d. The utility of an antibiogram is that it shows antimicrobial susceptibility trends in a localized population.

Fill in the Blank

The method that tin can decide the MICs of multiple antimicrobial drugs against a microbial strain using a single agar plate is called the ________.

Show Respond

The method that tin can determine the MICs of multiple antimicrobial drugs against a microbial strain using a single agar plate is called the Etest.

True/Simulated

If drug A produces a larger zone of inhibition than drug B on the Kirby-Bauer disk improvidence exam, drug A should ever be prescribed.

Retrieve about It

1. How is the data from a Kirby-Bauer disk diffusion exam used for the recommendation of the clinical utilise of an antimicrobial drug?
2. What is the difference between MIC and MBC?
3. Can an Etest be used to discover the MBC of a drug? Explicate.

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What Factors Influence The Size Of The Zone Of Inhibition Produced By A Chemical,

Source: https://courses.lumenlearning.com/microbiology/chapter/testing-the-effectiveness-of-antimicrobials/

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