antibiotic susceptibility testing practical wrap up Flashcards
What is the method of testing for AST by disk diffusion?
(Prep desk with chemgene)
1. Standard agar is MHA, in petir dish, label with microbe to be added, date and agar name on the outside of the base
2. Dip a sterile cotton swap in bacterial suspension, rotate swab around edge of tube to remove excess fluid
3. Inoculate the surface of the agar by streaking the swab at a low angle to the agar, repeat for a total of three times rating 60 degrees between each for full and even coverage, keep the lid as closed as possible over the agar during this process.
4. Go around the edge of the plate with the swab to pick up any excess liquid
5. Lid plates and allow to sit at room temp for 5-15 mins
6. PArtially remove lid and use sterile forceps to place antibiotic disk on surface of agar, gently press disk to ensure even contact with the agar surface.
7. Place as many disks as needed, spread out appropriately so that zone of inhibition will not overalpw with each other of the edge of the plate.
(Clean desk with chemgene)
8. Incubate at 37 degrees overnight
9. Measure diameter of zone on inhibition, larger zone indicates more susceptible, record value
10. Compare to zne diameter breakpoints for specific antibiotic to determine is susceptible, intermediate or resistant.
why is antimicrobial susceptibility testing important in clinical practise?
Ensure antibiotic choice is appropriate for patient-specific microbe, reduce mortality and morbidity, influence decisions in primary, secondary and tertiary care. Avoid treatment failure and unnecessary antibiotic use.
This can help prevent resistance spreading by ensuring only the most effective antibiotics are used.
Aids public health as can monitor for rates and patterns of resistance, this influences local and national antibiotic prescribing policy.
Used in the development of new antibiotics, to known potential indications of use.
In summary - key tool in antimicrobial stewardship.
What is antimicrobial susceptibility testing?
Laboratory procedure to identify which antimicrobial regime is effective against certain microbe cultures, often linked to a specific patient.
Allow accurate and rapid identification of resistance to antimicrobial drugs.
Can be used to identify the MIC of the drug, hence the concentration of antibiotic at which resistance begins.
What are the 3 main different methods used for antimicrobial susceptibility testing?
- Disk diffusion - antibiotic disk on agar with microbe culture, incubate measure zone of inhibition
- Agar dilution method - incorporate different concentrations normal by a dilution process of antimicrobial agent into an agar medium, then adding microbe and use spectrophotometer to measure growth via absorbance (more growth more absorbance)
- ATP bioluminescence assay - measure adenosine triphosphate produce by bacteria or fungi, quantify to estimate microbial population. Requieres D-luciferen to react with ATP producing oxylucifern with generates light which can be measured with a luminometer, add antibiotic and compare growth levels
What are the advantages and limitation of the disk diffusion method of measuring antimicrobial susceptibility?
+ easy to do as no specialist equipment needed
+ results are relatively quick only 24 hours wait.
+ can test many antibiotics at once
- Zones of inhibition may be difficult to see, not accurate as margin of error and subjectivity in human measuring of distance
- Results are different on different agars and culturing conditions, creating variability in results.
- Limited spectrum of antibiotics, newer antibiotics may not yet be available as disks so cant be included in testing.
What are the two different categories of methods used to test for AMR?
Phenotypic methods - inc disk diffusion, borth microdilution, agar dilution, gradient diffusion methods
Gentoypic methods - PCR to amplify and measure DNA sequences associated with antimicrobial genes, DNA sequencing, microarroar analysis.
What are the advantages and limitation of genotypic methods of assessing for AMS?
- expensive technologies
- Clarithromycin induced antimicrobial resistance - contains gene but not expressed, unable to differentiate between this in genetic testing
- May not identify new resistance genes that are not in sequences to look out for
+ Highly sensitivity for specific genes hence methods of resistance
+ Rapid, produce results within a few hours allowing for timely treatment
+ Can be automated, reducing the risk from human error
What are the advantages and limitations of agar dilution method for AMR testing?
+ provides quantitative results including the MIC, useful to have information on the conc not just the type of antibiotic used
+ Is highly reproducible across lots of laboratories, ensures accurate conclusions
- labour intensive in the preparation fo multiple agar plates/well plate/ different concentration of microbial agents, increases the time and effort involved in producing results
- result interpretation can be complex, may require specially trained staff.
How are the results of AST interpreted?
Measure result from specific practical
Often compare growth is standard chart range, use reference values to check is susceptible, intermediate or resistance to antibiotic.
How does AST guide the selection of appropriate antimicrobial therapy for a patient?
- Ensures microbe is susceptible to chosen antibiotics and avoiding ineffective antibiotics. Tailed to specific pathogen in patient.
- Guidance on dosing/conc of antibiotics to give a patient.
- Reducing the risk of patients developing antibiotic-resistant strains during treatment.
- Results from different patients with similar presentations can be collated over time, used to build up local and national pictures of resistance that can then be used to guide empirical treatment guidelines.
How does AST help in monitoring and managing antibiotic resistance?
- Shows resistance trends specific to microbe, may recognise new resistant strains
- Can collate data on a local and national level and temporally to understand have resistance varies geographically and how it has changed over time.
- Ensures patients are given the appropriate antibiotic, reducing occurrence of resistance
- Identify new resistance mechanisms, indicate novel method that needs further investigation by public health or government bodies.
What is AST quality control?
What are some methods?
Aims to find, reduce and correct deficencies in labs internal analytical process prior to the release of patient results in order to improve the quality and accuracy of results provided.
May include use of reference strains being tested alongside sample, compare reference to expected results ensure method if working correctly.
Use of standardized procedures to ensure results are reproducible
Why is AST quality control important?
- Ensures results are accurate hence treatment guidance is reliable and optimising patient care, this indicates the laboratory protocol and procedure are correct.
- Antimicroibial stewardship - if done correctly can limit antibiotic use and ensure only correct type is used.
- Important in research, ensures accurate results to influence conclusions on the effectiveness or implications of old/new antibiotic.
How does AST testing vary in different parts of the world?
Variation due to: laboratory capacity in technology and time, availability of resources such as antibiotic samples or agar wells, local epidemiology of resistance (areas with no resistance, test less as less concerned), presence and power of laboratory accreditation to deny or give license effect the amount of research lab going on.
High in USA, Europe - strong healthcare system, emphasises of antimicrobial stewardship and research funding.
Low in sub-saharan Africa and south Asia - struggling health care resources, infractsturuct and laboratory facilities. Antimirobial resistance is not a priority to them.
Discuss the global challenges in AST, especially in low resource settings?
Expensive technology, cultures and laboratory facilities to ensure testing is safe and accurate - limited funding
Lack the healthcare organisation to take responsibility for testing.
Lacks the fundraising potential of other health concerns such as cancer - difficult to raise money to fund projects
Lack of educated and trained personnel in order to carry out testing
Even if testing occurs - may have insecure supply of limited range of antibiotics so only able to prescribe what they have - worsen resistance
Other competing health priority such as COVID19 vaccine development or malaria treatment etc.
Lack of awareness or standardisation or AST procedure, struggle to compare results to other regions or results are of poor quality.
