LAB S18 Flashcards by Leanne Grace Fernandez

an in vitro measure to determine the susceptibility of microorganisms to one or several antimicrobial agents

Antimicrobial susceptibility testing (AST)

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Data from the evaluation of microorganisms’ antibiogram are reported in the form of

minimum inhibitory concentration (MIC)

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Latin for “within the glass”

In vitro

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Latin for “within the living”

in vivo

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compounds which act against microorganisms by blocking essential cellular processes

Antimicrobial agents

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what cellular processes are blocked by antimicrobial agents

cell wall synthesis
protein synthesis
DNA replication/transcription
cellular respiration

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antimicrobial agents that inhibit microbial growth are called

microbistatic

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antimicrobial agents that kill microorganisms are called

microbicidal

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the susceptibility profile of a microorganism

antibiogram

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lowest concentration of an antibiotic that inhibits visible growth of a microorganism in an in vitro system

Minimum Inhibitory Concentration

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Antimicrobial susceptibility tests are widely used for bacterial infections but are less frequently used for fungal, viral, and parasitic infections. T or F

True

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uses of antimicrobial susceptibility testing

As a chemotherapeutic guide
As an epidemiological surveillance tool
As a laboratory diagnostic tool

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pharmaceutical agents that include antibiotics (antibacterials), antifungal, antiviral, and antiparasitic drugs

antimicrobial drugs

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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.

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This result implies that therapy with the recommended dosage of a particular antibiotic is likely to be effective in eradicating the infection

susceptible

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indicates that the antibiotic in the appropriate dose has not
been shown to have a high likelihood of treatment success in clinical trials

resistant

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AST plays an essential role in guiding the clinician in the prescription of
antibiotics for treatment of an infection

AST as a chemotherapeutic guide

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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.

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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.

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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

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predicts the effectiveness of the antibiotic being tested against a particular bacterial species or strain.

Antimicrobial Susceptibility Testing

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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.

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In vitro susceptibility is a prerequisite for the _______ of an
antibiotic

in vivo efficacy

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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.

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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.

LAB S18 Flashcards

(80 cards)