Lab
9
|
Objectives: |
Reading: |
|
1. Antibiotic Susceptibility & MIC |
1. Tortora, Pages 549-550 |
|
2. Skin Specimen |
2. P: page 127; L: pages 93-94 |
|
3. Handout |
Antimicrobial Susceptibility Testing
When a microorganism has
been isolated from a patient specimen, the obvious question is ‘how can we
eliminate this organism’? The goal of
antimicrobial susceptibility testing is to predict the success or failure of
antibiotic therapy in the patient.
‘In the patient’ is
considered an in vivo (living) situation. Unfortunately, all tests are performed in vitro (literally
‘in glass tubes,’ it means outside of the patient). Consequently, results should be viewed in the context of the
patient’s clinical information and prior clinician experience with the selected
antimicrobial.
All antimicrobial susceptibility testing is
performed under standardized test conditions.
The results must be reproducible.
This means that, regardless of who performs the test, which lab the test
is performed in, the circumstances surrounding the test performance, or any
other variable, the test results will always be the same for that particular
sample.
Today we will look at two
types of antimicrobial susceptibility testing, the Kirby-Bauer disc diffusion
test and the minimal inhibitory concentration test.
Kirby-Bauer Test
A large agar plate is inoculated uniformly with a
standardized amount of bacteria.
‘Uniformly’ means that there is an equal amount of bacteria throughout
the entire plate. Some people call this
a “lawn.” ‘Standardized’ means that
there are policies and procedures in place that have been accepted by
microbiologists throughout the world as to the exact quantity of bacteria that
is to be used for the inoculum. This
quantity is consistently adhered to.
Following this, small filter paper disks (about 6 mm in
diameter), each of which has been impregnated with a standard amount of
antibiotic, are placed onto the agar plate.
The plate is incubated.
During incubation, the chemotherapeutic agents diffuse from the disks
into the agar. The concentration of the
agent is greatest, of course, closest to the disk and least at the furthest
distance from the disk.
Once incubation is completed, microbiologists measure zones
of inhibition, in mm, as an indicator of the effectiveness of the
agent. A zone of inhibition is a
circular area radiating from the disk in which growth of the microorganism has
been completely inhibited. The diameter
of this zone is measured and then compared to a standard table in order to
correctly interpret the results.
Antimicrobial susceptibility of the microorganism is defined
as the organism either being sensitive, intermediate, or resistant to the
chemotherapeutic agent.
- Sensitive
means that the organism is susceptible to the antibiotic. The implication to the clinician here
is that an infection caused by the strain of microorganism isolated by the
laboratory may be treated with the usual dosage of the tested
antimicrobial agent.
- Resistant means that the antibiotic does not inhibit the organism. The implication to the clinician here is than an infection caused by the strain of microorganism isolated by the laboratory will not respond appropriately to the normal dosage schedule for this particular antimicrobial agent.
- Intermediate defines the continuum of susceptibility and resistance. Antimicrobial susceptibility is not black and white—sensitive or resistant. There is a gray area that is best interpreted by the clinician, based on his experience with the drug and his patient’s clinical picture. Infections caused by organisms in this category may or may not respond to therapy with the tested agent.
To read a zone of inhibition for a particular antimicrobial, a metric
ruler is used. The ruler, above, is
divided into 20 centimeters. Each
centimeter is equal to 10 millimeters.
Zones of inhibition are measured in millimeters. Do not confuse the centimeter numbers with
the millimeter markings.
To make a reading,
- Place
the ruler across the widest part of each zone (the diameter)
- Count
the number of millimeters (mm) across the zone measures
- Record
that number under “DM” (diameter in mm) on the chart below
- Compare
your number to the interpretive chart below, and determine
whether your organism is sensitive (S), intermediate (I), or resistant (R)
to the antimicrobial
Kirby-Bauer Antibiotic Agar Diffusion Test and Broth
Dilution Test Results Table
|
Antibiotic (Abreviation) |
Escherichia coli |
Staphylococcus epidermidis |
Enterococcus faecalis |
Pseudomonas aeruginosa |
Klebsiella pneumoniae |
Serratia marcesens |
||||||
|
|
DM |
SIR |
DM |
SIR |
DM |
SIR |
DM |
SIR |
DM |
SIR |
DM |
SIR |
|
Amoxicillin/Clavulinic Acid (AMC) |
|
|
|
|
|
|
|
|
|
|
|
|
|
Ampicillin (AM) |
|
|
|
|
|
|
|
|
|
|
|
|
|
Carbenicillin (CB) |
|
|
|
|
|
|
|
|
|
|
|
|
|
Cefoxitin (FOX) |
|
|
|
|
|
|
|
|
|
|
|
|
|
Cephalothin (CR) |
|
|
|
|
|
|
|
|
|
|
|
|
|
Chloramphenicol (C) |
|
|
|
|
|
|
|
|
|
|
|
|
|
Gentamicin (GM) |
|
|
|
|
|
|
|
|
|
|
|
|
|
Imipenem (IPM) |
|
|
|
|
|
|
|
|
|
|
|
|
|
Moxalactam (MOX) |
|
|
|
|
|
|
|
|
|
|
|
|
|
Penicillin (P) |
|
|
|
|
|
|
|
|
|
|
|
|
|
Sulfoxasole (G) |
|
|
|
|
|
|
|
|
|
|
|
|
|
Vancomycin (VA) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Ampicillin Broth Dilution (MIC) |
|
|
|
|
|
|
||||||
Zone Diameter Interpretive Chart Based on Data Provided by
the
National Committee for Clinical Laboratory Standards (NCCLS)
|
Antimicrobial Agent |
Code |
Disc Potency |
Zone
Diameter Interpretive Standards (mm) |
||
Resistant
|
Intermediate |
Susceptible |
|||
|
Chloramphenicol (for non-Haemo-philus sp.) |
C-30 |
30 mg |
<12 mm |
13-17 mm |
>/= 18
mm |
Penicillin
(for
staphylococci) |
P-10 |
10 U |
</= 28
mm |
|
>/= 29
mm |
|
Penicillin (for enterococci) |
P-10 |
10 U |
</= 14
mm |
|
>/= 15
mm |
|
Penicillin (for enterococcal streptococci) |
P-10 |
10 U |
</=19
mm |
20-27 mm |
>/= 28
mm |
|
Streptomycin |
S-10 |
10 mg |
</= 11
mm |
12-14 mm |
>/= 15
mm |
|
Tetracycline (for most organisms) |
TE-30 |
30 mg |
</= 14
mm |
15-18 mm |
>/= 19
mm |
|
Trimethoprim |
TMP-5 |
5 mg |
</= 10
mm |
11-15 mm |
>/= 16
mm |
|
Amoxicillin/ Clavulinic Acid for staphylococci |
|
|
<19 mm |
|
>20 mm |
|
Amoxicillin/ Clavulinic Acid for all others |
|
|
<13 mm |
14-17 mm |
>18 mm |
|
Imipenem |
|
|
<13 mm |
14-15 mm |
>16 mm |
|
Moxalactam |
|
|
<14 mm |
15-22 mm |
>23 mm |
Minimum
Inhibitory Concentration
The minimum inhibitory concentration, better known as the MIC, is the lowest
concentration of an antimicrobial that visibly inhibits bacterial growth in a
given time period. It differs from the
Kirby-Bauer method in that it provides the clinician with a quantitative value,
rather than a qualitative value.
To determine the MIC for a particular antimicrobial,
- Serial
dilutions of each antibiotic are made
- A
standardized inoculum of the bacterial colony is placed into each of the
diluted tubes
- After
an overnight incubation, the tubes are examined for evidence of growth
- The MIC
is reported as the lowest concentration of the antimicrobial that inhibits
visible growth of the bacteria
In today’s lab, we will compare one antimicrobial, ampicillin, using
the MIC and the Kirby-Bauer methods.
The amipicillin was serially diluted out, from an original concentration
of 64 mg/mL to a final concentration of 0.125 mg/mL, using twofold dilutions.
On the chart on the previous page, note the minimum dilution of the
ampicillin at which growth of each microorganism was visibly inhibited.
Normal Skin Flora
Human skin is home to literally billions of microorganisms. Most are Gram positive bacteria that thrive
on sebum and sweat. Sebum, a product of
the sebaceous glands,
and sweat provide nutrients and moisture. When these bacteria break down the sebum, the product, fatty
acids, is inhibitory to most Gram negative bacteria. In addition to Gram positive bacteria, some fungi and viruses may
be found on skin.
In today’s lab, we will take skin samples (from ourselves) and streak
them onto mannitol salt agar (MSA) and blood agar. Recall that blood agar is both basic and differential, whereas
MSA is selective for halophiles such as Staphylococcus, which is Gram positive.
To take a skin culture, roll a moistened sterile swab over the skin
site that you wish to investigate. Then
roll the swab across the MSA and blood agar plates. We will examine the cultures next week (and sub-culture as
necessary).
Take Out Food for the Brain:
“Flesh-eating bacteria”
have been in the news recently. Is this
a new group of bacteria? The answer is
old group, but more virulent strain.
Necrotizing fasciitis, as this disease is known within the medical
field, is caused by Streptococcus pyogenes.
S. pyogenes is implicated in a variety of human diseases, including
strep throat, scarlet fever, and various skin conditions. Most infections of the skin that are caused
by S. pyogenes are usually secondary to a primary lesion caused by
another organism.
With necrotizing
fasciitis (literally ‘dying, inflamed soft tissue’), there may be some minor
trauma. Some cases occur following
surgery, especially abdominal surgery.
However, there are cases where there is no noticeable injury to the
skin.
The bacteria attack
the subcutaneous tissue (the soft tissue under the skin), which rapidly becomes
gangrenous. The infection can actually
be observed to move, and tissue destruction is known to occur at the rate of
two inches per hour. Once the tissue
has died, it must be removed. If caught
early, removal of flesh, subcutaneous tissue, and some fat may halt the spread
of the disease (along with antimicrobial therapy). However, limb amputation is often required, and victims may
succumb to respiratory failure, heart failure, or renal failure due to the
necrotizing toxin’s effects.
The necrotizing toxin
is thought to be a streptococcal erythrogenic pyrogenic toxin (SPE). ‘Erythrogenic’ means the toxin causes a
reddening (of the tissues involved), and ‘pyrogenic’ means the toxin induces
fever production. This is an example of
an exotoxin. Recall the origin of
exotoxins is from bacteriophages, viruses that infect bacteria.
Where does this
particular strain of bacteria come from?
Anywhere that Streptococcus pyogenes are found. S. pyogenes are not normal skin
flora, but they are present in the environment and can enter through an
abrasion, a puncture wound, or a cut.
Three conditions are
thought to be necessary for necrotizing fasciitis to occur:
- Contusion, abrasion, cut, or
opening in skin (idiopathic cases are reported, but rare)
- Come in contact with someone or
something having S. pyogenes
- Must be invasive strain of S.
pyogenes having SPE
Why might the human
immune response be unable to respond to the threat of S. pyogenes? You tell me.
Leg infected with S.
pyogenes,
resulting in necrotizing fasciitis.
Take Home Thought
Things aren’t always what they seem to be. Sometimes they are much worse.
Menu