Define the Minimum inhibitory concentration (MIC):
The minimum concentration that inhibits visible growth
What is the term for the minimal concentration to inhibit visible growth?
Minimum inhibitory concentration (MIC)
What is the definition for significant bactericidal activity?
Killing of three logs (99.9%) or greater
What is the term for killing of three logs or greater (i.e. 99.9%)
Significant bacterial activity
Minimum bactericidal concentration is defined as:
Lowest concentration with significant bactericidal activity
What is the term for the lowest concentration with significant bactericidal activity?
Minimum bactericidal activity
Frequency of resistant mutants is:
1 mutant per 10^6 to 10^9 viable bacterial cells
1 mutant per 10^6 to 10^9 viable bacteria cell represents what statistic?
Frequency of resistant mutants
What is a breakpoint?
A determining committee, such as the FDA
Define CLSI:
Clinical and Laboratory Standards Institute
Define EUCAST:
European Committee on Antimicrobial Susceptibility Testing
What categories of test results for susceptibility exist?
Susceptible
Intermediate
Resistant
Give examples of determining committees or Breakpoints:
FDA
CLSI
EUCAST
The FDA, EUCAST and CLSI all fall under what greater category?
Breakpoints or Determining committees
How do we detect resistance?
Susceptibility tests
Good susceptibility is a guarantee of success with a given antimicrobial agent (T/F)?
False
Susceptibility tests are not adequate (T/F)?
True, we need to optimize detection of resistance
Therapeutic success depends on what other factors other than susceptibility?
Penetration of drug at infection site
Immune status
Need for surgery
___________ is dependent on Immune status, penetration of drug at infection site and need for surgery?
Therapeutic success
Disk diffusion can be described as what type of measure of susceptibility?
Qualitative
Name a qualitative test for susceptibility:
Disk diffusion
Potency of drugs can be compared using qualitative means (T/F)?
False
What was discussed in class as not accomplishable via Qualitative testing?
Comparison of drug potency
What examples were given in class for Qualitative tests?
Measure of susceptibility
Comparison of drugs
Macrobroth dilution
Microbroth Dilution
Agar dilution
E-test
E-test, measure of susceptibility, drug comparisons, macrobroth dilution, microbroth dilution and agar test are all what general type test?
Quantitative
Describe the process of creating a Macrobroth dilution:
1-3 mL Volumes of broth
Dilute drug in 2 fold dilution to get to 0.06 ug/mL of the drug
Inoculate with 5E5 CFU/mL
Incubate for 18-24 hours
What is the inherent Error of the susceptibility test?
Inherent 2 fold error of MIC test
i.e. 2ug/ml can be 1-4 ug/ml
What is the Inoculum effect?
The inoculum in the MIC test is much lower than in a real infection
Does not account for pathogens production of antibiotic inactivating enzymes
What technical difficulties may arise in susceptibility tests?
Fuzzy or trailing endpoints
Difficult to interpret
What limitations are inherent in the MIC test?
Inherent error = inherent 2 fold error in dilutions
Inoculum effect = inoculation in MIC test is usually much lower than in vivo/does not account for pathogen production of antibiotic inactivating enzyme
Technical difficulties= diff. to interpret results
Macrobroth dilution is what general type of assay?
Quantitative
What are the advantages of a Macrobroth dilution?
Quantitative
Most conservative (gives the highest MIC)
Gold standard
Can measure Minimum Bactericidal concentration
What test is quantitative, conservative (giving the highest MICs), able to measure MBC and considered the Gold standard for susceptibility assays?
Macrobroth Dilution
What disadvantages are there for Macrobroth Dilution?
Labor intensive
Inoculums below 10^6 = missed mutants
What test is unable to detect inoculums below 1066 and is considered labor intensive?
Macrobroth Dilution
What is the dilution volume for Microbroth dilution?
0.1mL
Describe the Microbroth Dilution method:
0.1mL volumes
Dilute 2 fold to reach 0.06ug/mL
50,000 CFU/mL inoculum
Incubate 18-24 hours
Why is Microbroth considered less accurate than a Macrobroth?
The smaller the sample size the less accurate the results
Why is a Macrobroth Dilution considered more accurate than a Microborth?
Larger sample size equates to more accurate results
When accuracy is the most important component of your testing, what susceptibility assay would you choose, Microbroth or Macrobroth?
Macrobroth
Describe an agar dilution susceptibility test:
Spread plating of agar is performed on plates of varying concentration
What are the benefits of using an Agar dilution susceptibility test?
Quantitative
32 strains per drug plate
Easier than macrobroth
What susceptibility test is Quantitative, easier than macroborth and allows for 32 strains to be plated per plate?
Agar Dilution
What disadvantages exist for the Agar dilution susceptibility test?
Does not yield a minimum bactericidal concentration
MICs are lower than macrobroth
Lower inoculum
What test does not yield MBCs, has MICs that are lower than macrobroth and a low inoculum?
Agar Dilution
An E-test is similar to what other tests?
Agar dilution and disk diffusion
What susceptibility test is similar to an agar dilution and disk diffusion?
E-test
Describe the method for an E-test:
Spread a lawn of bacteria
Apply E-test strip
Incubate for 18-24 hours
What test requires a lawn of bacteria, an antibiotic gradient strip and an incubation period of 18-24 hours?
E-test
Describe the characteristics of an E-test strip:
Plastic strip coated with antibiotic gradient
What is the name for a strip coated with an antibiotic gradient?
E-test Strip
In the E-test what happens to the antibiotic during incubation?
Diffuses into the agar at rate dependent on the concentration
Describe how you read an E-test:
The point of the E-test strip that aligns with the lowest concentration point where bactericidal activity can be detected= MIC
What are the advantages of the E-test:
Quantitative
6 drugs can be tested concurrently
Easy
What susceptibility test allows for 6 drugs to be tested concurrently, is quantitative and easy to perform?
E-test
What are the disadvantages of the E-test?
Does not yield MBCs
Costly
Results are difficult to interpret
What susceptibility test has results that are difficult to interpret, does not produce MBC data and is costly?
E-test
Describe the method for Disk Diffusion:
Spread a lawn of bacteria
Apply antibacterial impregnanted disk
Incubate 18-24 hours
What test requires a lawn to be spread, antibacterial impregnated disks to be layed on the lawn and an incubation period of 18-24 hours?
Disk Diffusion
How is a disk diffusion test interpreted?
By measuring the zone of inhibition
What susceptibility test is interpreted by measuring the zone of inhibition?
Disk Diffusion
What variables control the zone of inhibition in the Disk diffusion test?
Temperature
Concentration of Drug disk
Inoculum size
Susceptibility of test organism
Temperature, Conc. Of drug, inoculum size and susceptibility of test organism are all variable factors for what susceptibility test?
Disk Diffusion
What are the advantages of disk diffusion?
Fast and easy to perform
Inexpensive
Many drugs can be tested concurrently
Drug interactions may be detected
New drugs are usually available for this test type
What is the disadvantage of the disk diffusion assay?
You cannot tell HOW sensitive the organism is from the zone size
What I easy, fast, inexpensive, able to determine the susceptibility of many drugs concurrently, can detect drug interactions and has new drugs available for testing?
Disk Diffusion
What are the advantages of automated susceptibility tests?
Rapid, accurate and cheap
Reduce workload
Very specific computer analyzed results
What are the disadvantages for automated susceptibility tests?
Algorithms may be inaccurate for certain organisms, drugs or inoculum concentrations
A negative result for a Molecular detection of resistance genes test means what?
Does not mean the organism is susceptible
What are the shortcomings of Molecular detection of resistance genes?
Genes that are too diverse may not be detected
What are the positive aspects of Molecular detection of resistance genes?
Potential for:
Rapid results
Accurate results
Highly specific results
What issue require resolution prior to routine use of Molecular detection of resistance gene testing?
Cost
Convenience
Preventative measures for over and understating resistance
Needs methods for detecting new resistance genes
Phenotypic tests for resistance have a broader scope than Molecular detection of resistance genes test (T/F)?
True
What is the point of Irreversibility?
The point beyond which treatment will have no impact on an outcome of an infection
What is the term for the point beyond which most appropriate treatment will have no impact on the outcome?
Point of irreversibility
What has been well studied in terms of the point of irreversibility?
Pneumonia (most well studied)
Sepsis
Meningitis
Neutropenia
Pneumonia, Sepsis, Meningitis and Neutropenia have all been studied (Pneumonia more so than the others) in terms of what characteristic?
Point of irreversibility
What differences in Rapid vs microdilution susceptibility methods were discovered by the Doern study?
The mortality rate was lower for those receiving a rapid susceptibility method
Fewer procedures were used on patients receiving rapid methods
Faster witch from empiric Rx
Why is early antibiotic administration more robust?
Attacks the pathophysiological process
What is the social impact of using Rapid susceptibility methocs?
Reduction of therapy cost
2.4 million Saved/year
Early isolation of patient=less unnecessary spread
How can we diminish the development of resistance?
