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 a clinician to change the choice of antibiotics from empiric therapy, which is when an antibiotic is selected based on clinical suspicion about the site of an infection and common causative bacteria, to directed therapy, in which the choice of antibiotic is based on knowledge of the organism and its sensitivities.
Sensitivity testing usually occurs in a medical laboratory, and uses culture methods that expose bacteria to antibiotics, or genetic methods that test to see if bacteria have genes that confer resistance. Culture methods often involve measuring the diameter of areas without bacterial growth, called zones of inhibition, around paper discs containing antibiotics on agar culture dishes that have been evenly inoculated with bacteria. The minimum inhibitory concentration, which is the lowest concentration of the antibiotic that stops the growth of bacteria, can be estimated from the size of the zone of inhibition.
Antibiotic susceptibility testing has been needed since the discovery of the beta-lactam antibiotic penicillin. Initial methods were phenotypic, and involved culture or dilution. The Etest, an antibiotic impregnated strip, has been available since the 1980s, and genetic methods such as polymerase chain reaction testing have been available since the early 2000s. Research is ongoing into improving current methods by making them faster or more accurate, as well as developing new methods for testing, such as microfluidics.
Uses
In clinical medicine, antibiotics are most frequently prescribed on the basis of a person's symptoms and medical guidelines. This method of antibiotic selection is called empiric therapy, and it is based on knowledge about what bacteria cause an infection, and to what antibiotics bacteria may be sensitive or resistant. For example, a simple urinary tract infection might be treated with trimethoprim/sulfamethoxazole. This is because Escherichia coli is the most likely causative bacterium, and may be sensitive to that combination antibiotic. However, bacteria can be resistant to several classes of antibiotics. This resistance might be because a type of bacteria has intrinsic resistance to some antibiotics, because of resistance following past exposure to antibiotics, or because resistance may be transmitted from other sources such as plasmids. Antibiotic sensitivity testing provides information about which antibiotics are more likely to be successful and should therefore be used to treat the infection.Antibiotic sensitivity testing is also conducted at a population level in some countries as a form of screening. This is to assess the background rates of resistance to antibiotics, and may influence guidelines and public health measures.
Methods
Once a bacterium has been identified following microbiological culture, antibiotics are selected for susceptibility testing. Susceptibility testing methods are based on exposing bacteria to antibiotics and observing the effect on the growth of the bacteria, or identifying specific genetic markers. Methods used may be qualitative, meaning that a result indicates resistance is or is not present; or quantitative, using a minimum inhibitory concentration to describe the concentration of antibiotic to which a bacterium is sensitive.There are many factors that can affect the results of antibiotic sensitivity testing, including failure of the instrument, temperature, moisture, and potency of the antimicrobial agent. Quality control testing helps to ensure the accuracy of test results. Organizations such as the American Type Culture Collection and National Collection of Type Cultures provide strains of bacteria with known resistance phenotypes that can be used for quality control.
Phenotypic methods
Testing based on exposing bacteria to antibiotics uses agar plates or dilution in agar or broth. The selection of antibiotics will depend on the organism grown, and the antibiotics that are available locally. To ensure that the results are accurate, the concentration of bacteria that is added to the agar or broth must be standardized. This is accomplished by comparing the turbidity of bacteria suspended in saline or broth to McFarland standards—solutions whose turbidity is equivalent to that of a suspension containing a given concentration of bacteria. Once an appropriate concentration has been reached, which can be determined by visual inspection or by photometry, the inoculum is added to the growth medium.Manual
The disc diffusion method involves selecting a strain of bacteria, placing it on an agar plate, and observing bacterial growth near antibiotic-impregnated discs. This is also called the Kirby-Bauer method, although modified methods are also used. In some cases, urine samples or positive blood culture samples are applied directly to the test medium, bypassing the preliminary step of isolating the organism. If the antibiotic inhibits microbial growth, a clear ring, or zone of inhibition, is seen around the disc. The bacteria are classified as sensitive, intermediate, or resistant to an antibiotic by comparing the diameter of the zone of inhibition to defined thresholds which correlate with MICs.Image:E-test Ngono.jpg|thumb|left|upright=0.9|Example of an Etest, which uses a plastic strip impregnated with an antibiotic at a range of concentrations
Mueller–Hinton agar is frequently used in the disc diffusion test. The Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing provide standards for the type and depth of agar, temperature of incubation, and method of analysing results. Disc diffusion is considered the cheapest and most simple of the methods used to test for susceptibility, and is easily adapted to testing newly available antibiotics or formulations. Some slow-growing and fastidious bacteria cannot be accurately tested by this method, while others, such as Streptococcus species and Haemophilus influenzae, can be tested but require specialized growth media and incubation conditions.
Gradient methods, such as Etest, use a plastic strip placed on agar. A plastic strip impregnated with different concentrations of antibiotics is placed on a growth medium, and the growth medium is viewed after a period of incubation. The minimum inhibitory concentration can be identified based on the intersection of the teardrop-shaped zone of inhibition with the marking on the strip. Multiple strips for different antibiotics may be used. This type of test is considered a diffusion test.
In agar and broth dilution methods, bacteria are placed in multiple small tubes with different concentrations of antibiotics. Whether a bacterium is sensitive or not is determined by visual inspection or automatic optical methods, after a period of incubation. Broth dilution is considered the gold standard for phenotypic testing. The lowest concentration of antibiotics that inhibits growth is considered the MIC.
Automated
Automated systems exist that replicate manual processes, for example, by using imaging and software analysis to report the zone of inhibition in diffusion testing, or dispensing samples and determining results in dilutional testing. Automated instruments, such as the VITEK 2, BD Phoenix, and Microscan systems, are the most common methodology for AST. The specifications of each instrument vary, but the basic principle involves the introduction of a bacterial suspension into pre-formulated panels of antibiotics. The panels are incubated and the inhibition of bacterial growth by the antibiotic is automatically measured using methodologies such as turbidimetry, spectrophotometry or fluorescence detection. An expert system correlates the MICs with susceptibility results, and the results are automatically transmitted into the laboratory information system for validation and reporting. While such automated testing is less labour-intensive and more standardized than manual testing, its accuracy can be comparatively poor for certain organisms and antibiotics, so the disc diffusion test remains useful as a backup method.Genetic methods
Genetic testing, such as via polymerase chain reaction, DNA microarray, and loop-mediated isothermal amplification, may be used to detect whether bacteria possess genes which confer antibiotic resistance. An example is the use of PCR to detect the mecA gene for beta-lactam resistant Staphylococcus aureus. Other examples include assays for testing vancomycin resistance genes vanA and vanB in Enterococcus species, and antibiotic resistance in Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli. These tests have the benefit of being direct and rapid, as compared with observable methods, and have a high likelihood of detecting a finding when there is one to detect. However, whether resistance genes are detected does not always match the resistance profile seen with phenotypic method. The tests are also expensive and require specifically trained personnel.Polymerase chain reaction is a method of identifying genes related to antibiotic susceptibility. In the PCR process, a bacterium's DNA is denatured and the two strands of the double helix separate. Primers specific to a sought-after gene are added to a solution containing the DNA, and a DNA polymerase is added alongside a mixture containing molecules that will be needed. If the relevant gene is present, every time this process runs, the quantity of the target gene will be doubled. After this process, the presence of the genes is demonstrated through a variety of methods including electrophoresis, southern blotting, and other DNA sequencing analysis methods.
DNA microarrays and chips use the binding of complementary DNA to a target gene or nucleic acid sequence. The benefit of this is that multiple genes can be assessed simultaneously.
Using magnetic nanoparticles studded with a beta-2-glycoprotein I peptide imitating a plasma protein, microbial pathogens could selectively be retrieved from blood culture specimens within hours, in a study published September 2024. Magnets are used to fish out the peptide-bacterial complex, followed by genetic testing.