Once a bacterium has been identified following microbiological culture,<unk> s are selected for susceptibility<unk> . Susceptibility<unk>  methods are based on exposing bacteria to<unk> s and observing the effect on the growth of the bacteria (phenotypic<unk> , or identifying specific genetic markers (genetic<unk> . Methods used may be qualitative, meaning that a result indicates resistance is or is not present; or quantitative, using a minimum inhibitory concentration (MIC) to describe the concentration of<unk>  to which a bacterium is sensitive. There are many factors that can affect the results of<unk> , including failure of the instrument, temperature, moisture, and potency of the antimicrobial agent. Quality control (QC)<unk>  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 From left to right: 0.5, 1, and 2 McFarland standards<unk>  based on exposing bacteria to<unk> s uses agar plates or dilution in agar or broth. The selection of<unk> s will depend on the organism grown, and the<unk> s that are available locally. To ensure that the results are accurate, the concentration of bacteria that is added to the agar or broth (the inoculum) 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 (most commonly an 0.5 McFarland standard) 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<unk> -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<unk>  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<unk>  by comparing the diameter of the zone of inhibition to defined thresholds which correlate with MICs. Example of an alt=Bacteria are growing on an agar plate upon which a strip containing varying concentrations of<unk> s has been placed. An elliptical zone without growth is present around areas with higher concentrations of the<unk> . Mueller–Hinton agar is frequently used in the disc diffusion test. The Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility<unk>  (EUCAST) 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<unk>  newly available<unk> s 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<unk> s 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<unk> s 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<unk> s. 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<unk> . The lowest concentration of<unk> s 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<unk> , or dispensing samples and determining results in dilutional<unk> . 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<unk> s. The panels are incubated and the inhibition of bacterial growth by the<unk>  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<unk>  is less labour-intensive and more standardized than manual<unk> , its accuracy can be comparatively poor for certain organisms and<unk> s, so the disc diffusion test remains useful as a backup method. Genetic methods Genetic<unk> , such as via polymerase chain reaction (PCR), DNA microarray, DNA chips, and loop-mediated isothermal amplification, may be used to detect whether bacteria possess genes which confer<unk>  resistance. An example is the use of PCR to detect the mecA gene for beta-lactam resistant Staphylococcus aureus. Other examples include assays for<unk>  vancomycin resistance genes vanA and vanB in Enteroccocus species, and<unk>  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<unk>  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 (for example, nucleotides and ions). 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. MALDI-TOF Matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) is another method of susceptibility<unk> . This is a form of time-of-flight mass spectrometry, in which the molecules of a bacterium are subject to matrix-assisted laser desorption. The ionised particles are then accelerated, and spectral peaks recorded, producing an expression profile, which is capable of differentiating specific bacterial strains after being compared to known profiles. This includes, in the context of<unk>  susceptibility<unk> , strains such as beta-lactamase producing E coli. MALDI-TOF is rapid and automated. There are limitations to<unk>  in this format however; results may not match the results of phenotypic<unk> , and acquisition and maintenance is expensive.
Antibiotic sensitivity testing
Microbiological culture
Blood culture
Sputum culture
Culture conversion
Digital contact tracing
Empiric therapy
Bacteriology