Susceptibility Testing Bacteria Flashcards
General approach to identification and susceptibility
-Early identification of specimens for likely organisms.
Later ID=worse outcomes.
Knowing organism and what AB we need to treat with.
-Guide initial antimicrobial therapy.
-Susceptibility testing to determine appropriateness of therapy.
Delay=failure and resistance.
-Monitor for efficacy and resistance throughout.
Study:
Bloodstream infections in patients in the hospital
Used adquate therapy and the risk of mortality is about 30%, inadequate therapy is 60%.
33% of S. aureus infections received initial inadequate therapy.
Diagnostic Paradigm
- Patient
- Diagnostic differential
- Collect specimens and cultures
- Serology (diagnostic titers, titer conversion); direct detection (direct staining-gram, tissues, fluids, antigen detection, toxin detection, molecular assays); culture (biochemical reactions to differentiate organisms, antisera-blood serum containing antibodies to a specific antigen, molecular methods, susceptibility)
Takes a long time to happen
Growing an organism for culture can take 7-80 hours to grow, then susceptibility testing which adds another day.
Minimum Inhibitory Concentraton (MIC)
Lowest concentration of given antimicrobial that will inhibit (visual) the patient’s organism from growing after 18-24 h incubation.
Not talking about the killing margin.
Indications for determination:
-Isolation of an organism with unpredictable susceptibility patterns.
Until 1990, routine susceptibility testing for S. pneumonia was not performed; macrolide and tetracycline resistance; important to screen how resistance is evolving in community and hospital.
Most send samples for routine surveillance to monitor resistance evolution.
Group A strep; universally susceptible to penicillin; no need to do susceptibility test because we know there is no resistance.
-Isolation of organisms of clinical significance, such as isolates from normally sterile sources (urine, CSF, blood, etc.); culture from a normally sterile site; send off for testing depending on setting; to see if what is appropriate therapy.
-Initial identification of what you deem to be appropriate therapy.
-See average of concentration; wide distribution (heterogeneity in organism MIC).
Organism population and MIC is reflecting most common MIC in that population.
Can see evolution-see certain population start to grow and then that is your resistant population.
Is resistance already present in the population or are we creating it?
Taking a portion that is already resistant and allowing it to overcome the patient and the rest of the population; regrowth of a population and this leads to resistant effects.
Minimum Bactericidal Concentration (MBC)
Lowest concentration of a given antimicrobial that will kill (99.9%) of the patients’ organism after 18-24 h incubation.
If you take the antibiotic away, the organism is dead and cannot regrow.
Determined following the performance of the broth dilution MICs.
Method:
-An aliquot (usually 5-10 micrometers) dilutions of the antimicrobial agents MIC are subcultured onto antibiotic-free agar.
-Plates are incubated for 18-24 hours at 35 celsius and then examined for growth.
-Lowest concentration of antibiotics able to kill > or equal to 99.9% of original inoculum is the MBC.
Will the organism grow if you take the antibiotic away?
Determination of MBC is not routinely performed procedure.
The test is not standardized between laboratories. May be determined for serious infections (meningitis, endocarditis, etc) or to help characterize investigational agents.
Tolerance: defined as a wide disparity between MIC and MBC (MIC/MBC ration > or equal to 16).
Can tolerate drug until goes away and then comes back to cause infection.
Tolerance expression rather than resistance.
Clinical significance is unknown.
Susceptibility breakpoints
Concentration that separates populations of microorganisms.
Obtained from microbiologic data, animal modeling (effect of organism), PK/PD modeling (computers, MIC is this, what is the probability of patients having successful therapy), and human clinical trials.
Classification:
-Susceptible (S): isolate may be treated by antimicrobial for recommended infections.
-Intermediate (I): infection may be treated with higher doses or drug concentration sites; can overcome the intermediated resistance; penicillin against S. pneumo.
-Resistant (R): infection by isolate not treatable
Nonsusceptible (NS): newer antimicrobials with few resistant strains; such little resistant out there, they do not know the breakpoint for what is resistant, but they know what is susceptible; treat it as resistant and would treat it as such by not using the AB.
Useful in determining predictive parameters of efficacy: AUC/MIC; T>MIC, etc.
AUC of the drug over a 24 h period.
T=time
Parameters used to predict an outcome all relating to the susceptibility of the organism.
Determining the level of drug that an institute deems an organism being susceptible or resistant; MIC number.
Example: MIC=1, do you call this susceptible or resistant?
Concentration that separates a population of organisms.
Setting the breakpoint
Definition: concentration of an antimicrobial that separates populations organisms.
Purpose: provides information to clinicians to select optimal antibiotic therapy; S, I, or R; which AB would be useful for a treatment.
May be set for drug classes (tetracyclines); can ahem a breakpoint for an entire class.
Cephalosporin; can have multi breakpoints for each individual antibiotic.
Depends on this and the individual organism.
Unique to each organism and antibiotic.
MICs
Animal models
In vitro models
Computer models (MonteCarlo modeling)
Human clinical data=
BREAKPOINT!
Guiding Committees for Breakpoints
FDA vs Clinical and Laboratory Standards Institute (CLSI)
-Breakpoint for the FDA may not match independent committee recommendation.
“This MIC (level of drug) is acceptable to treat with this AB.”
-FDA breakpoints often reflect those approved upon an NDA.
-CLSI meets yearly and establishes changes in breakpoints, which may differ from the FDA; breakpoints may change due to:
-Unless a company applies for a NDA change, the FDA breakpoint will not change.
-US laboratories are certified based on compliance with CLSI.
-Changing resistance patterns can change breakpoints.
-Patients dont do as well if the MIC is at this concentration…
Example: vancomycin against s. aureus; FDA breakpoint is 4, CLSI breakpoint is 2, want to change to 1.
What you see in the insert may not actually be what you use in the clinical setting.
The lowest number MIC looks like its the best; most potent.
Gemifloxacin, the most potent fluoroquinolone against strep.
Limitations: less than or equal to sign.
Does the drug get to the site of infection?
Who care about the MIC if the drug never gets into the urine with a UTI.
Give drug 5x per day
Toxicities.
Other interpretive Standards
More at a population level.
Percentiles
MIC50: concentration at which 50% of the organism population is inhibited by the selected antibiotic.
MIC90: concentration at which 90% of the organism population is inhibited by the selected antibiotic; often reported in surveillance studies and institutional susceptibilities, useful in comparing/tracking activity o antimicrobials.
This level of drug is inhibiting 50% of the organisms at that level or lower.
MIC50 and MIC90 that are the same is a good thing; potent and stable drug as you increase more the population.
Can show you resistance patterns in large population type of exposure.
Comparative activities of FQs
The antimicrobial with the lowest MIC50/MIC90 is not always the most active.
Other factors to consider:
-PK/PD relationship; can you achieve an appropriate concentration of that drug where you need it to affect that MIC.
-Tissue penetration
-Protein binding; 95% protein bound; only free drug is active against bacteria; low MIC and high protein binding, not a lot of free drug.
-Susceptibility breakpoints
Tetracyclines are universally the same in their breakpoints; differences for FQs and differences for cephs within the class.
Disk Diffusion (Kirby-Bauer Test)
-Qualitative test applicable to organisms that grow rapidly on artificial media.
Organism is streaked across surface of agar followed by application of antibiotic impregnated disks.
After 18-24 h incubation (normal) at 35 celsius, inhibition zones are measured (mm).
Size of zone correlates with susceptibility.
Results are expressed as S (sensitive), I (intermediate), or R (resistant) based on known repository or organisms compared to your unknown or sample from patient.
Advantages: speed, low cost and minimum labor; can do many assays on one platform; high throughput.
Disadvantage: no MBC, misinterpretation of new and rare resistance; not measuring cidality only inhibition.
Might have resistance thats subtley occurring in the population and you may not be able to detect it with this assay because it has not been validated against a number of new resistance mechanisms.
Drop a disk of AB on a plate and look at how large around that disk is inhibited.
If you mis treat and try to escalate back up broadly, it does not matter, the patient still has double risk of mortality.
Severe pneumonia, FQ bc active against strep.
Mild infection= Z pack.
Standards are always used; a lot of limitations to this.
Can do a lot with a small amount of space and test a lot of AB.
Do not get number here; you just get a number is it S or R.
Broth dilution
Quantitative test results expressed as mcg/ml.
Macrotube vs microtube
-Macrotube (old way of doing it): serial two fold dilutions of antimicrobial agents are made into growth medium followed be the addition of bacteria at a standard inoculum (~10^6 CFU/ml).
Tubes are examined for turbidity after 18-20 h incubated at 35 celsius.
The MIC is defined as the tube containing the highest dilution of antimicrobial inhibiting visual growth.
Getting the level of drug that is needed to inhibit bacterial growth.
Microtube: automated susceptibility testing; just ass sample to a plate and put in a machine to interpret 18-24 h later; same as macro tube, only in smaller volumes in plastic micrometer plates. Inoculum size and incubation conditions are the same; employed by many hospitals due to adaptability to automation (Microscan, Vitek, etc.)
-Advantgaes: ability to determine MBC and automated; is it killing or just inhibiting growth.
-Disadvantages: labor intensive and time consuming; difficult on large scales.
Can do different strains and antibiotics in a small amount needed.
Not really telling you if it is killed, just if it is inhibiting bacterial growth.
Different concentrations in each column; the level of drug that is needed to inhibit bacterial growth.
Automated Susceptibility Testing
Might not be able to test the antibiotic you want to test because it does not come as a commercial standard.
Reports susceptibility to up to 25 agents after 4-24 h of incubation:
-VTEK 1
-VTEK 2
-MicroScan WalkAway
-BD Phoenix
-Sensititer ARIS 2x
Companies have different panels available for different organisms; companies want other companies to put their drugs on the plate; we need test to determine results in patients; have to get on the plate.
Clinical Utility
Advantages:
-Reduced labor/time costs
-Reproducibility
-Data management
-Rapid results: most <12 hours; slow replication rates, this does not happen; most fastidious organisms, fast.
Disadvantages:
-Increased equipment costs
-Predetermined panels
-Inability to test all clinically relevant pathogens.
-Inaccurate detection of resistant phenotypes: VRE, ESBLs, Psudeomonas resistance, reduced glycopeptide susceptibility, inducible resistance (expose and then expresses that resistance, do not know there is resistance until treatment failure); cannot be detected reliably on these platforms.
Epsilometer Test (Etest)
Quantitative test expressed as mvc/ml:
Similar to disk diffusion, antibiotics diffuse from plastic strips into agar to interact with bacteria that have been streaked onto the agar plate.
Each plastic strike is impregnated with the antibiotic in a gradient fashion. The strip is placed directly onto the surface of the agar plate with standardized inoculum.
Plate is incubated or 18-24 h at 37 celsius.
A tear shaped zone of inhibition is formed and the MIC is read as the lowest point of intersection on the E-strip.
New antibiotic have na E test available; need an individual test to run each antibiotic; expensive.
Do not get some bactericidal concentration levels; only interpreting MIC.
Modified disk diffusion test.
Measure the level of drug that is inhibiting the growth where it touches the strip.
Micro level of susceptibility.
Only for one drug.
Advantages: Quantify MIC, easy to perform with high reproducibility, multiple antibiotics can be tested per plate.
Disadvantages: E strips are very expensive, MBC cannot be determined by this method.
Clinical important of MIC; Vancomycin vs MRSA
Can find data related to patient outcome based at MIC.
Even though it shows susceptibility; it has different levels of susceptibility impacting the patients outcome.
Within suscpetibiltiy range, it can still be different in patient outcome.
30% different in response rate for patients with MIC of 1 or 2, which are both within susceptible range.
Systematic review and meta analysis of all the data.
A lot of groups are done by pharmacy researchers; overall forest plot showing outcome of meta analysis.
Even though the organism is susceptible, there is a big different in if the organism is highly or moderately susceptible, high MIC vs low MIC.
Changing breakpoint-clinical outcomes do not match the MIC for the breakpoint; lowering it will better reflect the patient clinical response.
