Kirby-Bauer Antibiotic Testing
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The disk diffusion test (also known as the agar diffusion test, Kirby–Bauer test, disc-diffusion antibiotic susceptibility test, disc-diffusion antibiotic sensitivity test and KB test) is a
culture Culture ( ) is a concept that encompasses the social behavior, institutions, and Social norm, norms found in human societies, as well as the knowledge, beliefs, arts, laws, Social norm, customs, capabilities, Attitude (psychology), attitudes ...
-based
microbiology Microbiology () is the branches of science, scientific study of microorganisms, those being of unicellular organism, unicellular (single-celled), multicellular organism, multicellular (consisting of complex cells), or non-cellular life, acellula ...
assay used in
diagnostic Diagnosis (: diagnoses) is the identification of the nature and cause of a certain phenomenon. Diagnosis is used in a lot of different academic discipline, disciplines, with variations in the use of logic, analytics, and experience, to determine " ...
and
drug discovery In the fields of medicine, biotechnology, and pharmacology, drug discovery is the process by which new candidate medications are discovered. Historically, drugs were discovered by identifying the active ingredient from traditional remedies or ...
laboratories. In diagnostic labs, the assay is used to determine the susceptibility of bacteria isolated from a patient's infection to clinically approved antibiotics. This allows physicians to prescribe the most appropriate antibiotic treatment. In drug discovery labs, especially
bioprospecting Bioprospecting (also known as biodiversity prospecting) is the exploration of natural sources for small molecules, macromolecules and biochemical and genetic information that could be developed into commercialization, commercially valuable prod ...
labs, the assay is used to screen biological material (e.g. plant extracts, bacterial fermentation broths) and drug candidates for antibacterial activity. When bioprospecting, the assay can be performed with paired strains of bacteria to achieve dereplication and provisionally identify antibacterial
mechanism of action In pharmacology, the term mechanism of action (MOA) refers to the specific biochemical Drug interaction, interaction through which a Medication, drug substance produces its pharmacological effect. A mechanism of action usually includes mention o ...
. In diagnostic laboratories, the test is performed by inoculating the surface of an agar plate with bacteria isolated from a patient's infection. Antibiotic-containing paper disks are then applied to the agar and the plate is incubated. If an antibiotic stops the bacteria from growing or kills the bacteria, there will be an area around the disk where the bacteria have not grown enough to be visible. This is called a zone of inhibition. The susceptibility of the bacterial isolate to each antibiotic can then be semi-quantified by comparing the size of these zones of inhibition to databases of information on known antibiotic-susceptible, moderately susceptible and resistant bacteria. In this way, it is possible to identify the most appropriate antibiotic for treating a patient's infection. Although the disk diffusion test cannot be used to differentiate bacteriostatic and bactericidal activity, it is less cumbersome than other susceptibility test methods such as broth dilution. In drug discovery labs, the disk diffusion test is performed slightly differently than in diagnostic labs. In this setting, it is not the bacterial strain that must be characterized, but a test extract (e.g. a plant or microbial extract). The agar plate is therefore inoculated with a bacterial strain of known phenotype (often an ATCC or NCTC strain), and disks containing the test extract are applied to the surface (see
below Below may refer to: *Earth *Ground (disambiguation) *Soil *Floor * Bottom (disambiguation) *Less than *Temperatures below freezing *Hell or underworld People with the surname * Ernst von Below (1863–1955), German World War I general * Fred Belo ...
). Zone of inhibition sizes cannot be used as a semi-quantitative measure of antibacterial potency because different extracts contain molecules with different diffusion characteristics (different
molecular size A molecule is a group of two or more atoms that are held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions that satisfy this criterion. In quantum physics, organic chemistry, ...
s, hydrophilicities etc.). Zone of inhibition sizes can be used for the purpose of dereplication though. This is achieved by testing each extract against paired strains of bacteria (e.g. streptomycin-susceptible and -resistant strains to identify streptomycin-containing extracts). Paired strains (e.g. wild type and
target Target may refer to: Warfare and shooting * Shooting target, used in marksmanship training and various shooting sports ** Bullseye (target), the goal one for which one aims in many of these sports ** Aiming point, in field artille ...
overexpressing strains) can also be used to identify antibacterial mechanism of action.


History

Agar diffusion was first used by
Martinus Beijerinck Martinus Willem Beijerinck (, 16 March 1851 – 1 January 1931) was a Dutch microbiologist and botanist who was one of the founders of virology and environmental microbiology. He is credited with the co-discovery of viruses A virus i ...
in 1889 to study the effect of
auxins Auxins (plural of auxin ) are a class of plant hormones (or plant-growth regulators) with some morphogen-like characteristics. Auxins play a cardinal role in coordination of many growth and behavioral processes in plant life cycles and are essenti ...
on bacterial growth. However, the method has been developed, refined and standardized by many scientists and scientific organizations over the years including George F. Reddish,
Norman Heatley Norman George Heatley OBE (10January 19115January 2004) was an English biologist and biochemist. He was a member of the team of Oxford University scientists who developed penicillin. Heatley developed the back-extraction technique for efficient ...
, James G. Vincent, Alfred W. Bauer, William M.M. Kirby, John C. Sherris, Hans Martin Ericsson, the
World Health Organization The World Health Organization (WHO) is a list of specialized agencies of the United Nations, specialized agency of the United Nations which coordinates responses to international public health issues and emergencies. It is headquartered in Gen ...
, the
Clinical and Laboratory Standards Institute The Clinical and Laboratory Standards Institute (CLSI) is a volunteer-driven, membership-supported, not-for-profit, standards development organization. CLSI promotes the development and use of voluntary laboratory consensus standards and guidelin ...
, the Swedish Reference Group for Antibiotics, the
Deutsches Institut für Normung ' (DIN; in English language, English, the German Institute for Standardisation) is a Germany, German non-profit organization and acting as national organization for standardization. DIN is the German International Organization for Standardizat ...
, the
British Society for Antimicrobial Chemotherapy The British Society for Antimicrobial Chemotherapy (BSAC) is a UK-based multi-professional organisation committed to preventing infectious diseases and tackling the growing threat of drug-resistant infections – one of the top global public heal ...
and others.


Principle

A pure bacterial culture is suspended in saline, its turbidity is standardized, and it is swabbed uniformly across an agar plate. An antibiotic- or extract-impregnated filter paper disk is then placed on the surface of the agar. The disk constituent(s) diffuse from the filter paper into the agar. The concentration of these constituents will be highest next to the disk and will decrease as the distance from the disk increases. If the antibiotic or extract is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is the zone of inhibition. In general, larger zones of inhibition correlate with lower
minimum inhibitory concentration In microbiology, the minimum inhibitory concentration (MIC) is the lowest concentration of a chemical, usually a drug, which prevents visible ''in vitro'' cell growth, growth of bacteria or Fungus, fungi. MIC testing is performed in both diagnosti ...
s (MICs) of antibiotic or extract for that bacterial strain. An exception to this is when molecules of the antibiotic or extract are large or hydrophobic because these diffuse through the agar slowly.


Standard method


Agar plate and inoculum preparation

All aspects of the Kirby–Bauer procedure are standardized to ensure consistent and accurate results. Because of this, a laboratory must adhere to these standards. The media used in Kirby–Bauer testing must be
Mueller–Hinton agar Mueller Hinton agar is a type of growth medium used in microbiology to culture bacterial isolates and test their susceptibility to antibiotics. This medium was first developed in 1941 by John Howard Mueller and Jane Hinton, who were microbiologis ...
at only 4 mm deep, poured into either 100 mm or 150 mm Petri dishes. The pH level of the agar must be between 7.2 and 7.4. Bacterial inoculum is prepared by diluting a broth culture to match a 0.5 McFarland turbidity standard, which is equivalent to approximately 150 million
cells Cell most often refers to: * Cell (biology), the functional basic unit of life * Cellphone, a phone connected to a cellular network * Clandestine cell, a penetration-resistant form of a secret or outlawed organization * Electrochemical cell, a d ...
per mL.


Inoculation and incubation

Using
aseptic technique Asepsis is the state of being free from disease-causing micro-organisms (such as pathogenic bacteria, viruses, pathogenic fungi, and parasites). There are two categories of asepsis: medical and surgical. The modern day notion of asepsis is deri ...
,
broth Broth, also known as bouillon (), is a savory liquid made of water in which meat, fish, or vegetables have been simmered for a short period of time. It can be eaten alone, but it is most commonly used to prepare other dishes, such as soups ...
culture Culture ( ) is a concept that encompasses the social behavior, institutions, and Social norm, norms found in human societies, as well as the knowledge, beliefs, arts, laws, Social norm, customs, capabilities, Attitude (psychology), attitudes ...
of a specific organism is collected with a
sterile Sterile or sterility may refer to: *Asepsis, a state of being free from biological contaminants * Sterile (archaeology), a sediment deposit which contains no evidence of human activity *Sterilization (microbiology), any process that eliminates or ...
swab. In the case of
Gram negative bacteria Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelope consists of ...
, excess liquid is removed from the swab by gently pressing or rotating it against the inside of the tube. The swab is then streaked across a Mueller–Hinton agar plate to form a bacterial lawn. To obtain uniform growth, the agar plate is streaked with the swab in one direction, rotated 120° and streaked again, rotated another 120° and streaked again. Using an antibiotic disk dispenser, disks containing specific antibiotics are then applied to the plate. This must be done within 15 minutes of inoculation. Flame-sterilized forceps are used to gently press each disk onto the agar and ensure it is attached. Plates are then incubated overnight, usually at a temperature of 35 °C. Plates must be incubated within 15 minutes of applying antibiotic disks.


Test media modifications

Certain bacteria require addition of 5% solution of mechanically defibrinated horse blood and β-NAD (MH-F agar). The following table shows media requirements of commonly tested microorganisms:


Quality control

To ensure veracity of test results,
quality control Quality control (QC) is a process by which entities review the quality of all factors involved in production. ISO 9000 defines quality control as "a part of quality management focused on fulfilling quality requirements". This approach plac ...
methods must be used. In order to monitor the performance of the test, special bacterial strains are used as positive or negative control. When efficacy of β-lactamase is tested, special strains that exhibit β-lactam resistance are used. Additionally, specific media are used to test certain antibiotics. For example, when testing
co-trimoxazole Trimethoprim/sulfamethoxazole, sold under the trade names Bactrim, Cotrim (a short form of the British Approved Name, Co-trimoxazole) and Septra, among others, is a fixed-dose combination antibiotic medication used to treat a variety of bac ...
susceptibility, media with excess
thymine Thymine () (symbol T or Thy) is one of the four nucleotide bases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine ...
and
thymidine Thymidine (nucleoside#List of nucleosides and corresponding nucleobases, symbol dT or dThd), also known as deoxythymidine, deoxyribosylthymine, or thymine deoxyriboside, is a pyrimidine nucleoside, deoxynucleoside. Deoxythymidine is the DNA nuc ...
are recommended. The following table lists commonly used quality control strains in the disk diffusion method:


Alternate methods

Several variations of the disk diffusion method have been developed including the Oxford penicillin cup and
Etest Etest (previously known as the Epsilometer test) is a way of determining antimicrobial sensitivity by placing a strip impregnated with antimicrobials onto an agar plate. A strain of bacterium or fungus will not grow near a concentration of anti ...
methods used in hospital diagnostic laboratories, and the well diffusion, cylinder diffusion and bioautography methods used in drug discovery and development laboratories.


Oxford penicillin cup method

Disks containing increasing antibiotic concentrations are placed on a seeded bacterial lawn on the agar surface and plates are incubated. Zone sizes are measured from the edge of the disk to the end of the clear zone. Interpretation is more complicated in mixed susceptibility populations. These are plotted as linear dimensions or squares of distances as a function of the natural logarithm of antibiotic concentration in the disks. The MIC is determined from the zero intercept of a linear regression fit through the data. The intercept itself is the logarithm of the MIC. The slope of the regression line is related to the diffusion coefficient of that particular antibiotic in the agar.


EUCAST Rapid Antibiotic Susceptibility Test (RAST)

The RAST method serves as a fast means of ascertaining antibiotic susceptibility and was created as a modification of the classic disk diffusion test. It allows to shorten the time of incubation to 16-20 hours. Test scores are read after 4, 6, 8 and 16-20 hours. Compared to the standard method, RAST does not give distinct zones of inhibition within such a short timespan (all bacteria except for ''S. pneumoniae'' have a chance of being possible to read after 6 hours higher than 90%). As for quality control strains, they are diluted 1:1 000 000 and defibrinated horse or sheep blood is added. Special RAST breakpoint tables should be used when interpreting the results due to method calibration differences. Validated quality control strains include: ''E. coli'' ATCC 25922, ''P. aeruginosa'' ATCC 27853, ''S. aureus'' ATCC 29213, ''E. faecalis'' ATCC 29212, ''S. pneumoniae'' ATCC 49619.


Screening for antibiotic resistance mechanisms in RAST

RAST allows for rapid determination of possible antibiotic resistance in tested cultures. It allows to check for ESBL and/or carbapenemase producing ''E. coli'' and ''K.'' pneumoniae, using cefotaxime/ceftazidime (after 4 hours) and meropenem (after 6 hours) respectively. However, these results are not quantitative and should be used only for screening in routine medical tests. In clinical trials, RAST method led to significant improvements in predicting efficacy of antibiotic therapy.


Disk pre-diffusion method

Disks containing antibiotics are placed on an uninoculated Mueller-Hinton agar plate and incubated for 2 hours. Then, they are removed and the bacterial suspension previously prepared using broth microdilution is applied and another disk with a different antibiotic is placed precisely in the same place as the previous one. After incubation for 16-20 hours results are correlated with the first antibiotic's MIC values. An example of the pre-diffusion method is testing ''in vitro'' efficacy of ceftazidime/avibactam (primary disks) in terms of aztreonam (secondary disks).


Antifungal drug testing

Disk diffusion method can be used to test susceptibility to antifungals.


Bioautography

Comparing to classical disk-based methods, bioautography utilises
thin-layer chromatography Thin-layer chromatography (TLC) is a chromatography technique that separates components in non-volatile mixtures. It is performed on a TLC plate made up of a non-reactive solid coated with a thin layer of adsorbent material. This is called the sta ...
to separate constituents of the tested mixture. Then, the TLC plate can be either placed on the inoculated agar and be allowed to diffuse into it (contact bioautography) or be covered with microbe-containing broth (direct bioautography). Then, the sample is incubated and zones of inhibitions are measured. Alternatively, the TLC plate can be covered with molten agar in the agar overlay bioautography. To visualise zones of inhibitions in direct bioautography, reagents that detect
dehydrogenase A dehydrogenase is an enzyme belonging to the group of oxidoreductases that oxidizes a substrate by reducing an electron acceptor, usually NAD+/NADP+ or a flavin coenzyme such as FAD or FMN. Like all catalysts, they catalyze reverse as well as ...
activity are used (e.g., tetrazolium salts, which are converted by microbial dehydrogenases into chromogenic formazans).


Other images

File:Martinus Willem Beijerinck.png, Agar diffusion was first used in 1889 by
Martinus Beijerinck Martinus Willem Beijerinck (, 16 March 1851 – 1 January 1931) was a Dutch microbiologist and botanist who was one of the founders of virology and environmental microbiology. He is credited with the co-discovery of viruses A virus i ...
. File:Agar Diffusion Method 2.jpg, A close-up look at the results of an agar diffusion test File:Serratia marcescens - antibiogram.jpg, An antibiogram of ''
Serratia marcescens ''Serratia marcescens'' () is a species of bacillus (shape), rod-shaped, Gram-negative bacteria in the family Yersiniaceae. It is a facultative anaerobe and an opportunistic pathogen in humans. It was discovered in 1819 by Bartolomeo Bizio in Pa ...
''. Each disk is labelled with the antibiotic it contains (e.g. AMC30, 30 μg
amoxicillin/clavulanic acid Amoxicillin/clavulanic acid, also known as co-amoxiclav or amox-clav, sold under the brand name Augmentin, among others, is an antibiotic medication used for the treatment of a number of bacterial infections. It is a combination consisting of ...
) File:Antibiotic Sensitivity Testing Plate reader.jpg, Automated culture plate reader to measure inhibition zone diameters


See also

*
Antibiotic sensitivity testing Antibiotic sensitivity testing or antibiotic susceptibility testing is the measurement of the susceptibility of bacteria to antibiotics. It is used because bacteria may have resistance to some antibiotics. Sensitivity testing results can allow ...
*
Double-disk diffusion test A double-disk diffusion test is a kind of disk diffusion test (to test for the effectiveness of an antimicrobial agent a disk infused with it is placed on a cultivated agar dish of bacteria to see if the antimicrobial agent in the disk inhibits fu ...
*
Etest Etest (previously known as the Epsilometer test) is a way of determining antimicrobial sensitivity by placing a strip impregnated with antimicrobials onto an agar plate. A strain of bacterium or fungus will not grow near a concentration of anti ...


References


Notes

{{Clinical microbiology techniques Microbiology techniques Antimicrobial resistance