Chapter 13 (MLS)

Antimicrobial susceptibility testing
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Why is antimicrobial susceptibility testing performed on isolated bacteria?
To determine which antimicrobial agents might be effective in treating infections caused by the bacteria- ONLY bacteria that are thought to cause infections should be tested
What is one of the major challenges in clinical microbiology?
is the identification of the bacteria that are the causes of infections
Susceptibility testing is usually performed by either disk diffusion or a dilution (minimal inhibitory concentration [MIC]) method
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What is the Clinical and Laboratory Standards Institute (CLSI)?
They are a publisher/ update that describe the standards that are done in the laboratory
What are some factors to consider when determining whether testing is warrented?
- the body site from which the organism was isolated
- the presence of other bacteria and the quality of the specimen from which the organism was grown
-the host's status
Body Site
susceptibility tests are NOT done on bacteria that are isolated from a body site that is a normal inhibitant of the bacteria.
You do not test them because that could confuse the physician when trying to treat a patient
Presence of other bacteria and quality of specimen
the isolate of an organism in pure culture is less likely to represent contamination than a mixed culture.
EX: more than 2 species at a greater than 10^5 CFU per mL isolated from the urine suggests that the urine is contamination and this may suggest susceptibility testing. HOWEVER a pure culture of e.coli at 10^5 per mL would likely represent a true infection and would be tested
Host Status
In immunosuppressed patients the normal biota that resides healthy individuals will infect them. Leaving the clinicians to test the normal biota on them. Also patients who are allergic to penicillin and who have a group A B-hemolytic streptococcal infection, erythromycin is the drug of choice, and B-hemolytic streptococci are occasionally resistant to this agent
Approx. 50 antimicrobial agents are in use presently for treating bacterial agents.
Each lab must determine which agents are appropriate for routine testing against various organisms in its certain setting. Representatives from different drug prescribers provide the input regarding the clinical utility of various agents in an institution.
The antimicrobial packets should be consulted for information concerning dosage amounts
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Generally a laboratory will define a battery of 10-15antimicrobial agents for routine testing against the Enterobacteriaceae, Psuedomonas spp, nonfastidious gram- bacteria.
Sometimes a separate battery is performed for urine isolates, representing drugs appropriate for treating UTIs
Often the bacteria being isolated is unknown, sometimes the testing of certain antimicrobial agents maybe inappropriate for reporting.
A drug should not be indiscriminately reported bcause results may be misleading. Some drugs may appear active against certain species in vitro but are inappropriate for clinical use. The final decision should be made once the bacteria being isolated is known
As a general guideline it is suggested that within a particular antimicrobial class, primary group A agents be reported first and that secondary Group B agents be reportedonly is one of the following conditions exists:
the isolate is resistant to the primary agents
the patient cannot tolerate the primary agents
the infection has not responded to the primary agents
a secondary agent would be a better clinical choice for the infection
the patient has organisms isolated from another site, and a secondary agent might be useful for treating both organisms
What is inoculum preparation?
It is one of the most critical steps in any susceptibility test. prepared by adding cells from 4-5 isolated colonies of similar colony morphology to a broth medium and then allowing them to grow to the log phase. Having 4-5 colonies rather than a single colony are selected to minimize the possibility of testing a colony that might have been derived from a susceptible mutant.
What are McFarland Turbidity Standards?
It is the most widely used method of inoculum standardization.Prepared by adding specific volumes of 1% sulfuric acid,and 1.175% barium chloride to obtain barium sulfate solution with a specific optical density.
The number of bacteria tested must be standardized regardless of the susceptibility method used. False results can occur if to little of bacteria were tested
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To standardize the inoculum, the inoculated broth or direst suspension is vortexed thoroughly, then under lighting is it positioned side by side with the .5 McFarland tube against a white card containing several horizontal black lines.
The turbidity is compared by looking at the black lines- it is dense if you can barely see the black lines compared to the McFarland tube.You then dilute the solution with saline or sterile broth. If it is too light you add more organisms and reincubated.
Dilution antimicrobial agents susceptibility test methods are used to determine the MIC the lost concentration of antimicrobial agent required to inhibit the growth of bacterial isolate
Once the MIC is determined, the organism is interpreted as either, nonsusceptible, susceptible, intermediate, or resistant
Antimicrobial stock solutions used in MIC must be prepared from reference standard antimicrobial powders, not from the pharmaceutical preparations admin, to patients
Broth dilution tests performed in test tubes are referred to as Broth-Macrodiltion MIC or tube-dilution MIC tests
Broth-Microdilution MIC testing uses plastic trays with 80-100 wells and .1mL are put into each well
the breakpoint panel is when only one or a few concentrations of each antimicrobial agent are tested on a single panel. The breakpoint is the term applied to the concentration of an antimicrobial agent that coincides with a susceptible or intermediate MIC break point for a particular drug.
What does it mean when a bacteria is susceptible?
it means that there is no growth in either of the 2 wells
What does it mean when a bacteria is intermediate
there is low concentration but no growth in the high concentration and resistant isolate grows in both cells
what does trailing involve?
it involves heavy growth at lower concentrations followed by one or more wells that show greatly reduced growth in the form of a small button or a light haze
Skipped wells involve growth at higher concentrations and no growth at one or more of the lower concentrations. This may occur as a result of contamination, improper concentrations of the antimicrobial
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establishing zone diameter interpretive breaking points
the amount of a drug at a specific location in the medium is unable to inhibit the growth of the test organism and a zone of inhibition is formed.
Zones of Inhibition is used to determine whether the bacteria is nonsusceptible, suseptible, intermediate, and resistant
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When reading the plates the quality control plates should be read before reading the patients to see if there were any mistakes.
If the growth is satisfactory then you measure the inhibition zone
Streptococcus pneumoniae and Sstreptococcus species
require a more nutritious medium for antimicrobial susceptibility. Broth tests are done on Mueller-Hinton medium that is supplemented with lysed horse blood
Factors to consider when determining whether testing is warranted:
body site
presence of other bacteria and quality of specimen
host status
Selecting antimicrobial agents for testing and reporting
selection of test batteries
reporting of susceptibility test results
Traditional antimicrobial susceptibility test methods
inoculum preparation
McFarland Turbidity standard
inoculum standardization
Dilution susceptibility testing methods
principle
antimicrobial stock solutions
Broth Macrodilution tests
Breakpoint MIC panels
trailing growth and skipped wells
agar dilution tests
Disk Diffusion tests
principle
establishing zone diameter interpretive breakpoints
test performance(disk storage, inoculation and incubation,reading plates and test interpretation)
Modified methods for testing slow growing or fastidious bacteria
-streptococcus pneumoniae and streptococcus app.
-haemophilus influenzae and Haemophilus parainfluenzae
-Neisseria gonorrhea and N. meningiditis
-anaerobes
-infrequently encountered or fastidious bacteria
-potential agents of bioterrorism
Additional organism and antimicrobial agent testing concerns
-detection of oxacillin resistance in Staphylococci
-vancomycin resistance or diminished susceptiblity in Staphylococcus aureus
-Inducible Clindamycin resistancec in Staphylococci
-Enterocci
-detection of high level aminoglycoside resistance in enterocci
-detection of vancomycin resistance in enterococci
There are many automated systems that read the tests for you
Automated instrument systems:
-BD phoenix system
-microscan walkaway SI
TReK ARIS
VVITEK1, VITEK 2, and VITEK 2 compact
Nonautomated antimicrobial susceptibility test method
Etest
Interpretation of in Vitro antimicrobial susceptibility test results

methods of detecting antimicrobial inactivating enzymes -B lactamase tests
Quality control of antimicrobial susceptibility tests

selecting an antimicrobial susceptibility test
Minimum bactericidal concentration (MCB) test -controlling test variable, interpretation concerns

time-kill assays
Synergy tests

serum bactericidal test

molecular probed fro identifying determinants of antimicrobial resistance
Minimum Bactericidal concnetratipn
measurement of antimicrobial agents in serum and body fluids
-biological assys
-immunoassays
-chromatographic assays
we are losing the war against bacteria
because of the emergence of microbial resistance
What are the 3 variables in the expression and transfer of bacteria resistance
location-
chromosomal- genetic expression, stability, often constitutive
extrachromosomal- plasmids easily mobilized for transfer from cell to cell
transposon- can move genetic material between chrmomsome and plasmid

transfer-
conjugation- either plasmids (R-factor) or transposons
transduction- transfer by bacteriophage
transformation- direct transfer of DNA between compatible species

expression-
constitutive- produced w or w/o exposure to stimulus
inductible- produced only after exposure to stimulus
constitutive-inducible- produced at low level w/o stimulus; production greatly increased after
What is enzymatic cleavage?
breaks it down
what is altered receptors (binding proteins)
cannot bind
altered permeability
changes cell wall (influx, efflux)
bypass of inhibitions
stopping metabolic pathways so there is no end product
Procedures in antimicrobial susceptibility testing
treat immuno-compromised patient differently tan healthy patient
make sure patients have infections before helping
grouping of recommended antimicrobial agents for testing and reporting
group
A- primary testing and report
B- primary test report selectively
C- supplemental testin and selective testin
D- supplemental testing of urinary tract infections
surrogate antibiotics
a antibiotic can be used for many different drugs
Minimum Inhibitory concentration MIC
the lowest concentration that inhibits growth
low MIC does not need a lot of antibiotics to kill it
the testing are accurate just not fast
Factors Positively affected by rpid determination of antimicrobial susceptibility in pathogenic bacteria
1 mortality rate
2 number of lab studies performed
3 number of imaging procedures
4 days of intubation
5 days spent in ICU
6 time to alteration of antimicrobial therapy
7 total cost of hospitalization
Etest
a strip with multiple [] on an infected plate- this test combines the broth dilution test with the MIC test
We have a rapid test for detecting B-lactamase
a disk test you put the colony on a disk and it breaks the disk down and turns it red
Quality Control
very important
-use QC strains for susceptibility testing- this will give us the exact result you need in which you can compare to make sure YOU are getting the correct answers
- you have to make sure its in the right percentage to be able to report it to the physician
-25% of tests done are the QC tests done in the hospital
Things that can go wrong
-inoculum
-Media- formulation, cation [], thymidine content, pH
-Incubation- atmosphere, temperature and length of time
Just because the bacteria is inhibited in a test doesn't mean its killed- streak a plate to test it
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MBC- minimum bacteriocidal concentration
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Autonomy, antagonism and synergism
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serum bacteriocidal test
to see if the antimicrobial is killing bacteria
measurement of antimicrobial agents in serum and body fluids
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Molecular probes for identifying determinants of antimicrobial resistance
detecting the gene that makes it resistant with a DNA probe