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.[1]
Sensitivity testing usually occurs in a
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 (PCR) 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
Antibiotic sensitivity testing is also conducted at a population level in some countries as a form of screening.[4] This is to assess the background rates of resistance to antibiotics (for example with methicillin-resistant Staphylococcus aureus), and may influence guidelines and public health measures.[4]
Methods
Once a bacterium has been identified following microbiological culture, antibiotics are selected for susceptibility testing.[5] Susceptibility testing methods are based on exposing bacteria to antibiotics and observing the effect on the growth of the bacteria (phenotypic testing), or identifying specific genetic markers (genetic testing).[6] 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 antibiotic to which a bacterium is sensitive.[6]
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.
Phenotypic methods
Testing based on exposing bacteria to antibiotics uses agar plates or dilution in agar or broth.[9] The selection of antibiotics will depend on the organism grown, and the antibiotics that are available locally.[5] 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)[10] has been reached, which can be determined by visual inspection or by photometry, the inoculum is added to the growth medium.[11][10]
Manual
The
Mueller–Hinton agar is frequently used in the disc diffusion test.[14] The Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) provide standards for the type and depth of agar, temperature of incubation, and method of analysing results.[11] 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.[5] Some slow-growing and fastidious bacteria cannot be accurately tested by this method,[5] while others, such as Streptococcus species and Haemophilus influenzae, can be tested but require specialized growth media and incubation conditions.[17]
Gradient methods, such as Etest, use a plastic strip placed on agar.[5] 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.[5] The minimum inhibitory concentration can be identified based on the intersection of the teardrop-shaped zone of inhibition with the marking on the strip.[5] Multiple strips for different antibiotics may be used.[5] This type of test is considered a diffusion test.[18]
In agar and broth dilution methods, bacteria are placed in multiple small tubes with different concentrations of antibiotics.[14] Whether a bacterium is sensitive or not is determined by visual inspection or automatic optical methods, after a period of incubation.[5] Broth dilution is considered the gold standard for phenotypic testing.[14] The lowest concentration of antibiotics that inhibits growth is considered the MIC.[5]
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.
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Multitarget microbial panel for automatic sensitivity testing. A small amount of the bacteria to be tested is placed in each well, each of which has the ingredients for a separate test.
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Microbial panels loaded into an instrument used for automated antibiotic sensitivity testing of each well.
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A laboratory worker reviews sensitivity results displayed on the screen of the automated analyzer.
Genetic methods
Genetic testing, such as via polymerase chain reaction (PCR), DNA microarray, and loop-mediated isothermal amplification, may be used to detect whether bacteria possess genes which confer antibiotic resistance.[9][23] An example is the use of PCR to detect the mecA gene for beta-lactam resistant Staphylococcus aureus.[9] Other examples include assays for testing vancomycin resistance genes vanA and vanB in Enteroccocus species, and antibiotic resistance in Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli.[9] These tests have the benefit of being direct and rapid, as compared with observable methods,[9] and have a high likelihood of detecting a finding when there is one to detect.[24] However, whether resistance genes are detected does not always match the resistance profile seen with phenotypic method.[9] The tests are also expensive and require specifically trained personnel.[25]
Polymerase chain reaction is a method of identifying genes related to antibiotic susceptibility.
DNA microarrays and chips use the binding of complementary DNA to a target gene or nucleic acid sequence.[9] The benefit of this is that multiple genes can be assessed simultaneously.[9]
MALDI-TOF
Matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) is another method of susceptibility testing.[6] This is a form of time-of-flight mass spectrometry, in which the molecules of a bacterium are subject to matrix-assisted laser desorption.[26] 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.[26] This includes, in the context of antibiotic susceptibility testing, strains such as beta-lactamase producing E coli.[9] MALDI-TOF is rapid and automated.[9] There are limitations to testing in this format however; results may not match the results of phenotypic testing,[9] and acquisition and maintenance is expensive.[25]
Reporting
Bacteria are marked as sensitive, resistant, or having intermediate resistance to an antibiotic based on the minimum inhibitory concentration (MIC), which is the lowest concentration of the antibiotic that stops the growth of bacteria. The MIC is compared to standard threshold values (called "breakpoints") for a given bacterium and antibiotic.
Clinical practice
Ideal antibiotic therapy is based on determining the causal agent and its antibiotic sensitivity. Empiric treatment is often started before laboratory microbiological reports are available. This might be for common or relatively minor infections based on clinical guidelines (such as
Specimens for antibiotic sensitivity testing are ideally collected before treatment is started.
When antibiotic sensitivity testing is completed, it will report the organisms present in the sample, and which antibiotics they are susceptible to.[27] Although antibiotic sensitivity testing is done in a laboratory (in vitro), the information provided about this is often clinically relevant to the antibiotics in a person (in vivo).[35] Sometimes, a decision must be made for some bacteria as to whether they are the cause of an infection, or simply commensal bacteria or contaminants,[27] such as Staphylococcus epidermidis[36] and other opportunistic infections. Other considerations may influence the choice of antibiotics, including the need to penetrate through to an infected site (such as an abscess), or the suspicion that one or more causes of an infection were not detected in a sample.[1]
History
Since the discovery of the beta-lactam antibiotic penicillin, the rates of antimicrobial resistance have increased.[37] Over time, methods for testing the sensitivity of bacteria to antibiotics have developed and changed.[25]
Alexander Fleming in the 1920s developed the first method of susceptibility testing. The "gutter method" that he developed was a diffusion method, involving an antibiotic that was diffused through a gutter made of agar.[25] In the 1940s, multiple investigators, including Pope, Foster and Woodruff, Vincent and Vincent used paper discs instead.[25] All these methods involve testing only susceptibility to penicillin.[25] The results were difficult to interpret and not reliable, because of inaccurate results that were not standardised between laboratories.[25]
Dilution has been used as a method to grow and identify bacteria since the 1870s, and as a method of testing the susceptibility of bacteria to antibiotics since 1929, also by Alexander Fleming.[25] The way of determining susceptibility changed from how turbid the solution was, to the pH (in 1942), to optical instruments.[25] The use of larger tube-based "macrodilution" testing has been superseded by smaller "microdilution" kits.[5]
In 1966, the
The Etest was developed in 1980 by Bolmstrӧm and Eriksson, and MALDI-TOF developed in 2000s.[25] An array of automated systems has been developed since and after the 1980s.[25] PCR was the first genetic test available and first published as a method of detecting antibiotic susceptibility in 2001.[25]
Further research
Additional research is focused at the shortcomings of current testing methods. As well as the duration it takes to report phenotypic methods, they are laborious, have difficult portability and are difficult to use in resource-limited settings, and have a chance of cross-contamination.[25]
As of 2017, point-of-care resistance diagnostics were available for
Quantitative PCR, with the view of determining the percent of a detected bacteria that possesses a resistance gene, is being explored.[9] Whole genome sequencing of isolated bacteria is also being explored, and likely to become more available as costs decrease and speed increases over time.[9]
Additional methods explored include microfluidics, which uses a small amount of fluid and a variety of testing methods, such as optical, electrochemical, and magnetic.[9] Such assays do not require much fluid to be tested, are rapid and portable.[9]
The use of fluorescent dyes has been explored.[9] These involve labelled proteins targeted at biomarkers, nucleic acid sequences present within cells that are found when the bacterium is resistant to an antibiotic.[9] An isolate of bacteria is fixed in position and then dissolved. The isolate is then exposed to fluorescent dye, which will be luminescent when viewed.[9]
Improvements to existing platforms are also being explored, including improvements in imaging systems that are able to more rapidly identify the MIC in phenotypic samples; or the use of bioluminescent enzymes that reveal bacterial growth to make changes more easily visible.[25]
Bibliography
- Burnett D (2005). The Science of Laboratory Diagnosis. Chichester, West Sussex, England Hoboken, NJ: Wiley. OCLC 56650888.
- Ford, M (5 June 2019). Medical Microbiology. Oxford University Press. ISBN 978-0-19-881814-4.
- Mahon C, Lehman D, Manuselis G (2018). Textbook of Diagnostic Microbiology (6 ed.). Elsevier Health Sciences. ISBN 978-0-323-48212-7.
- McPherson, RA; Pincus, MR (2017). Henry's Clinical Diagnosis and Management by Laboratory Methods (23 ed.). Elsevier Health Sciences. ISBN 978-0-323-41315-2.
References
- ^ PMID 21282489.
Once microbiology results have helped to identify the etiologic pathogen and/or antimicrobial susceptibility data are available, every attempt should be made to narrow the antibiotic spectrum. This is a critically important component of antibiotic therapy because it can reduce cost and toxicity and prevent the emergence of antimicrobial resistance in the community
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- ^ Mahon 2018, p. 95.
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- ^ a b Mahon 2018, p. 273.
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- ^ "WHO | World Health Organization". WHO. Archived from the original on October 19, 2014. Retrieved 2020-09-03.
The most common methods utilized are the disk diffusion susceptibility test method (also known as Kirby-Bauer)
- ^ ISBN 9781683672807.
- ^ Ford 2019, p. 61.
- ^ Mahon 2018, pp. 278–9.
- ^ Mahon 2018, pp. 279–82.
- ^ Burnett 2005, p. 169.
- ^ McPherson 2017, p. 1157.
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- ^ McPherson 2017, pp. 1157–8.
- ^ Ford 2019, p. 64.
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- ^ McPherson 2017, p. 1154.
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- ^ "Medical Definition of ANTIBIOGRAM". www.merriam-webster.com. Retrieved 2020-07-05.
- ^ a b "Antimicrobial Stewardship: Antibiogram | Wisconsin Department of Health Services". www.dhs.wisconsin.gov. 2021-08-09. Retrieved 2023-11-21.
- ^ Kirby-Bauer Disk Diffusion Susceptibility Test Protocol Archived 26 June 2011 at the Wayback Machine, Jan Hudzicki, ASM
- ^ Burnett 2005, p. 167.
- ^ Burnett 2005, pp. 135–144.
- ^ Burnett 2005, p. 168.
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- ^ Burnett 2005, p. 166.
- ^ "Diagnostics Are Helping Counter Antimicrobial Resistance, But More Work Is Needed". MDDI Online. 2018-11-20. Archived from the original on 2018-12-02. Retrieved 2018-12-02.
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External links
- "About Antibiograms (Antimicrobial Susceptibilites of Selected Pathogens)" (PDF). Minnesota Department of Health.