LAB S18 Flashcards
an in vitro measure to determine the susceptibility of microorganisms to one or several antimicrobial agents
Antimicrobial susceptibility testing (AST)
Data from the evaluation of microorganisms’ antibiogram are reported in the form of
minimum inhibitory concentration (MIC)
Latin for “within the glass”
In vitro
Latin for “within the living”
in vivo
compounds which act against microorganisms by blocking essential cellular processes
Antimicrobial agents
what cellular processes are blocked by antimicrobial agents
- cell wall synthesis
- protein synthesis
- DNA replication/transcription
- cellular respiration
antimicrobial agents that inhibit microbial growth are called
microbistatic
antimicrobial agents that kill microorganisms are called
microbicidal
the susceptibility profile of a microorganism
antibiogram
lowest concentration of an antibiotic that inhibits visible growth of a microorganism in an in vitro system
Minimum Inhibitory Concentration
Antimicrobial susceptibility tests are widely used for bacterial infections but are less frequently used for fungal, viral, and parasitic infections. T or F
True
uses of antimicrobial susceptibility testing
- As a chemotherapeutic guide
- As an epidemiological surveillance tool
- As a laboratory diagnostic tool
pharmaceutical agents that include antibiotics (antibacterials), antifungal, antiviral, and antiparasitic drugs
antimicrobial drugs
antimicrobial drugs often act within the host and subsequently damage the host, which is an important principle of selective toxicity. T or F
False. They often act within the host without damaging the host, which is an important principle of selective toxicity.
This result implies that therapy with the recommended dosage of a particular antibiotic is likely to be effective in eradicating the infection
susceptible
indicates that the antibiotic in the appropriate dose has not
been shown to have a high likelihood of treatment success in clinical trials
resistant
AST plays an essential role in guiding the clinician in the prescription of
antibiotics for treatment of an infection
AST as a chemotherapeutic guide
If the susceptibility profile is predictable, it is still necessary to do testing on an isolate. T or F
False. if the susceptibility profile is predictable, it is not necessary, in most
cases, to do testing on an isolate.
When is it not necessary to do a testing on an isolate
– Streptococcus pyogenes is usually susceptible to penicillin
– Anaerobes are susceptible to metronidazole
– Klebsiella pneumoniae is generally ampicillin-resistant
– Proteus species are generally resistant to nitrofurantoin and
tetracyclines.
Testing should be limited to those isolates that are definite pathogens, why?
testing of isolates that are “contaminants” or “normal flora” is
expensive and time-consuming and may lead to unnecessary
administration of antibiotics
predicts the effectiveness of the antibiotic being tested against a particular bacterial species or strain.
Antimicrobial Susceptibility Testing
AST is intended to be a guarantee that a particular agent or drug combination will be effective in therapy. T or F
False. AST is intended to be a guide for the
clinician, not a guarantee that a particular agent or drug combination will be effective in therapy.
In vitro susceptibility is a prerequisite for the _______ of an
antibiotic
in vivo efficacy
advantages of epidemiological surveillance tool
- aid to monitor susceptibility patterns of bacterial isolates
- aid in initiating appropriate empiric antibiotic therapy
- used to monitor emergence of acquired resistance.
bacterial pathogens of Community-acquired pneumonia (CAP)
- streptococcus pneumoniae
- haemophilus influenzae
- chlamydophila pneumoniae
- mycoplasma pneumoniae
- moraxella catarrhalis
Senior High Cool MM
Penicillin remains the standard drug of choice in the treatment of
low-risk CAP in patients without comorbid illness.
False. amoxicillin remains the standard drug of choice in the treatment of
low-risk CAP in patients without comorbid illness.
more than 10-20% of isolates of group A streptococcus (S. pyogenes) are
susceptible to bacitracin. T or F
False. more than 99% of isolates of group A streptococcus (S. pyogenes) are susceptible to bacitracin
a very small percentage of isolates of group B streptococcus and 10% to 20% of isolates of groups C and G streptococcus are also susceptible. T or F
True
Agar disk diffusion susceptibility tests became standardized in the United States based on the work of
Kirby, Bauer and coworkers
Principle of agar disk diffusion susceptibility
Agar disk diffusion method relies on the diffusion of antimicrobial agent released from an impregnated paper disk through the agar. As soon as the antimicrobial agent-impregnated disk comes in contact with the moist agar surface, water is absorbed into the filter paper and the antimicrobial agent
diffuses into the surrounding medium.
used routinely for testing common, rapidly growing, and certain fastidious bacterial pathogens because of its ease of use and lower cost compared to other methods.
agar disk diffusion method
random molecular motion
diffusion. high concentration to low concentration
important note
The rate of extraction of the antimicrobial agent out of the disk is
greater than its outward diffusion into the medium, so
that the concentration immediately adjacent to the
disk may exceed that in the disk itself.
As the distance from the disk increases, however, there is a logarithmic
reduction in the antimicrobial agent concentration.
When a critical cell mass of bacteria is reached, the inhibitory
activity of the antimicrobial agent is overcome and
bacterial growth occurs. T or F
True
The points at which the critical cell mass is reached appear as
a sharp circle with a margin of bacterial growth, with the middle of
the disk forming the center of the circle
The concentration of diffused antimicrobial agent at this interface of growing and inhibited bacteria is known as
critical concentration
-approximates the minimum inhibitory concentration (MIC) obtained in dilution tests
The sizes of zones of growth inhibition vary with the pharmacologic characteristics of different antimicrobial agent.
True
the zone size of one agent cannot be compared to the zone size of
another agent acting on the same organism. However, for any one agent, the zone size can be compared to a standard, provided that all conditions are controlled.
True
Standards for the practice of microbiology are published by
Clinical and Laboratory Standards Institute (CLSI)
covers the standards for agar disk diffusion method used to determine the in vitro antimicrobial susceptibility of bacteria that grow aerobically, and can be modified to test certain fastidious bacterial pathogens.
CLSI document M02
It describes how to perform the agar disk
diffusion and the criteria for quality control testing
CLSI document M02
presents tables that represent the most current information for drug selection, interpretation, and quality control using the procedures standardized in M02, M07, and M11
presents CLSI document M100
Standards for antimicrobial susceptibility of bacteria that grow aerobically using dilution methods.
M07
Standards for antimicrobial susceptibility of bacteria that grow anaerobically.
M11
Interpretative categories of the Agar Disk Diffusion Susceptibility Test
- Susceptible
- Resistant
- Intermediate
- Susceptible dose dependent (SDD)
- Nonsusceptible
Implies that the isolates are inhibited by the usually
achievable concentrations of antimicrobial agent when the
dosage recommended to treat the site of infection is used
resulting in likely clinical efficacy
Susceptible (S)
-A category defined by zone diameters at or above (or MIC at
or below) the susceptible breakpoint
Implies that isolates are not inhibited by the usually
achievable concentrations of the agent with normal dosage
schedules, or specific microbial resistance mechanisms are
likely and clinical efficacy of the agent against the isolate has
not been reliably shown in treatment studies.
Resistant (R)
-A category defined by zone diameters at or below (or MICs
at or above) the resistant breakpoint
Implies clinical efficacy in body sites where the drugs are
physiologically concentrated or when a higher than normal
dosage of the drug can be used. This category also includes
a buffer zone which should prevent small, uncontrolled,
technical factors from causing major discrepancies in
interpretations especially for drugs with narrow
pharmacotoxicity margins
Intermediate (I)
-A category defined by zone diameters (or MICs) within the
intermediate range that approach usually attainable blood
and tissue levels and for which response rates may be lower
than for susceptible isolates
A category defined by a breakpoint that implies that
susceptibility of an isolate is dependent on the dosage
regimen that is used in the patient
Susceptible dose dependent (SDD)
This should not be used when describing organism/drug
category with intermediate or resistant interpretive
categories.
Nonsusceptible (NS)
Isolates for which the zone diameters below (or MICs above) the value indicated for the susceptible breakpoint should be reported as
nonsusceptible
Variables of AST
- Inoculum
- test medium
- antimicrobial disks
- incubation
- reading and interpretation of results
Variables of the test found in the inoculum
- Purity
- Age
- Density
How to counter variabilities in the PURITY of inoculum
- pure culture should be sampled to minimize variation in the bacterial population
- select well-isolated colonies of the same morphological type
- get 3-5 same-looking colonies to minimize the possibility of testing susceptible colonies only and missing the resistant mutants
How to counter variabilities in the AGE of inoculum
-Bacteria to be tested must be in the logarithmic phase of
growth
-Broth culture method of inoculum preparation
-Colony suspension method of inoculum preparation
The inoculum is prepared from broth culture (usually
Trypticase Soy Broth, TSB) that has been incubated for 2-6 hours
Broth culture method of inoculum preparation
It is used for nonfastidious organisms (except
staphylococci) when fresh (18-24 hour) colonies are
not available
Broth culture method of inoculum preparation
formerly called growth method
Broth culture method of inoculum preparation
formerly called growth method
Broth culture method of inoculum preparation
formerly called direct colony suspension method
Colony suspension method of inoculum preparation
The inoculum is made by directly suspending 18- to 24-hour-old colonies from nonselective media (such as blood agar) in broth or saline
Colony suspension method of inoculum preparation
is used with most organisms if the time does not permit
incubation for 2 to 6 hours.
Colony suspension method of inoculum preparation
It is recommended for testing the fastidious organisms:
- Haemophilus influenzae
- Haemophilus parainfluenzae
- Neisseria gonorrhoeae
- Neisseria meningitidis
- streptococci
- for testing staphylococci to detect methicillin
(oxacillin) resistance.
Colony suspension method of inoculum preparation
Aging and autolysing bacteria may not produce confluent
lawn of bacterial growth on AST plate, for they tend to be more
susceptible
True
How to counter variabilities in DENSITY of the inoculum
-The TSB culture must contain a concentration of bacteria that
approximates the 0.5 McFarland turbidity standard,
approximately 1-2 x 108 colony-forming units
(CFUs) per milliliter
A lower inoculum concentration (too light)
may lead to
larger zone of inhibition;
higher inoculum concentrations (too dense) may lead to
smaller zone of inhibition
Use the inoculum suspension within 10 minutes
after adjusting the turbidity.
False. Use the inoculum suspension within 15 minutes
after adjusting the turbidity.
Variables in the Test Medium
- Type
- pH
- Depth
- Moisture
is considered the best medium for
the routine AST of non-fastidious bacteria
Mueller-Hinton Agar (MHA)
Why is Mueller-Hinton Agar (MHA) considered the best medium for
the routine AST of non-fastidious bacteria
-it shows acceptable batch-to-batch reproducibility for
AST.
- it is low in inhibitors that affect sulfonamides,
trimethoprim, and tetracycline susceptibility test results.
- it supports satisfactory growth of most non-fastidious
pathogens.
- a substantial amount of data and experience has been
collected about AST performed with this medium.
MHA has very low concentrations of divalent cations but is
adjusted (cation-adjusted MHA, CAMHA) to physiologic
concentrations ____________
during production
(20 to 35 mg/L Mg2+ and 50 to 100 mg/L Ca2+)
Mg2+ and Ca2+ affects
results of aminoglycoside and tetracycline tests with
Pseudomonas aeruginosa.
Excess cation content
reduces zone sizes,
low cation content may result in
unacceptably
large zone of inhibition
if there are variations in divalent cations, ________ must be used
MHA that is low in thymidine or thymine content (< 50 ug/ml)
Excessive amounts of thymidine or thymine can
reverse the
inhibitory effect of sulfonamides and trimethoprim thus yielding
smaller and less distinct zones or no zone at all. Enterococci are
particularly affected.
The MHA should have a pH between ______ at room
temperature.
7.2 and 7.4
If the pH <7.2
Erythromycin = smaller zone of inhibition Tetracyclines = larger zone of inhibition
check the pH of Test Medium by
-macerating enough agar to submerge the tip of a pH
electrode
- allowing a portion of agar to solidify around the tip of
a pH electrode in a beaker or cup
- using a properly calibrated surface electrode.
