1

Antimicrobial Susceptibility Testing

Learning Objectives:
Upon completion of this unit of instruction and lecture, the student
will be able to:
1. Explain the standardized procedure for each of the following
antimicrobial susceptibility tests, including all key components:
Kirby-Bauer diffusion testing
Micro-dilution testing
Beta-lactamase testing
2. Interpret results of the antimicrobial susceptibility tests listed in
Objective 1, including the use of standardized interpretative
criteria.
3. Recognize sources of error in antimicrobial susceptibility
testing, applying appropriate corrective action when necessary.
4. Evaluate the acceptability of antimicrobial test results based on
data of an organism’s characteristic antimicrobial profile.
5. Interpret special methods used when performing antimicrobial
susceptibility testing on the following organisms, including the
appropriate antimicrobial(s) used:
Methicillin-resistant Staphylococcus aureus (MRSA)
Enterococcus sp.
Enterobacteriaceae extended-spectrum beta-lactamase
producers
Streptococcus pneumoniae
Laboratory Learning Objectives:
Upon completion of this unit of instruction and laboratory
experience, the student will be able to:
1. Perform Kirby-Bauer susceptibility testing using standardized
procedures.
2. Perform beta-lactamase testing.
3. Interpret results of the antimicrobial susceptibility tests using
of standardized interpretative criteria.
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ANTIMICROBIAL AGENTS
Introduction
Antimicrobial agents include naturally occurring antibiotics,
synthetic derivatives of naturally occurring antibiotics (semi-
synthetic antibiotics) and chemical antimicrobial compounds
(chemotherapeutic agents).
Antimicrobial agents can be grouped by their mode of action, i.e.
their ability to inhibit the synthesis of the cell wall, cell membrane,
enzymatic activities, proteins and nucleic acids of bacteria.
The primary role of antimicrobial agents in microbiology bench:
To provide information in which physicians can diagnose and
treat diseases.
To identify specific pathogens from clinical specimen in order
to assist epidemiologists and other workers as well as the
patient in identification of infections. This is followed by
selection of an appropriate chemotherapy.

Definition
Antimicrobial agents are chemical substances that inhibit growth
of microorganisms or cause death of microorganisms.
There are three types of Antimicrobial agents:
Disinfectants.
Antiseptics.
Chemotherapeutic agents.

1. Chemotherapeutic agents.
These are chemical substances that have lethal effects or have
inhibitory effects on microorganisms but can be used for
therapeutic purposes at low concentrations. This means that they
have therapeutic effect on human or animal tissues. They can be
divided into two categories:
 Simple compounds.
These are chemotherapeutic agents that can be obtained by
laboratory synthesis e.g. drugs like Trimethoprim (septrin),
Sulphanomides, and Isoniazid etc.
 Produced by living organisms.
These are chemotherapeutic agents produced from living
organisms and are active against other living organisms e.g.
 3
Antibiotics like penicillin, Streptomycin, Polymixin B and
Cephalosporin etc.

Classification of Antibiotics
Antibiotics are classified based on the action they exhibit on
bacteria. These are:
 Bacteristatic drugs.
These are drugs when in their usual dosage (low
concentration) can inhibit growth and prevent active
multiplication of bacteria e.g. Chloramphenicol, Tetracycline
and Erythromycin.
 Bactericidal drugs.
These are drugs when in their usual dosage (low
concentration) can kill bacteria. They are more effective than
Bacteristatic drugs e.g. Penicillin, Glycopeptides, Nalidixic
acid, Cephalosporin and Aminoglycosides (Gentamycin,
Kanamycin, Amikacin and Neomycin Streptomycin etc).
Mode of action of antibiotics.
Most of the Antimicrobial agents function in one of the four major
ways. These are:
1. Inhibition of bacterial cell wall peptidoglycan synthesis e.g.
Penicillins (Amphicillin, Amocycillin, Penicillin G, Benzyl
penicillin etc).
Cephalosporin (Cefuroxime, Cefotaxime etc).
Glycopeptides (Vancomycin etc).

2. Inhibition of bacterial protein synthesis e.g.

Amino glycosides (Gentamycin, Streptomycin, Kanamycin
etc).
Macrolides e.g. Erythromycin.
Lincosamides e.g. Clindamycin.
Chloramphenicol
Tetracyclines e.g. Tetracycline and Doxycline.
3. Inhibition of bacterial nucleic acid synthesis e.g.
Quinolones e.g. Nalidixic acid, Ciprofloxacin and Norfloxacin
etc.
Sulphanomides and Trimethoprim e.g. Co-trimoxazole etc.
Metronidazole
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4. Damage of permeability of the bacterial cytoplasmic membrane
by the drugs e.g. Polymixin B.
5. Interference with the bacterial enzymatic activities.
Other antibacterial agents include: Nitrofurantoin etc.
6. Antimycobacterial agents e.g. Ethambutol, Rifampicin,
Isoniazid etc.
7. Antifungal agents e.g. Amphotericin B and Nystatin,
Imidazole, triazole, Griseofulvin.
8. Antiviral agents’ e.g.
Acyclovir (against herpes group of viruses).
Zidovudine (AZT) (inhibits HIV replication)
Tribavirin (ribavirin0
Interferon (inhibits replication of hepatitis B and C viruses).

Uses of antibiotics in the laboratory.

 They can be in-co-operated as selective agents in culture
media e.g. Thayer Martin agar medium whereby drugs:
 Vancomycin is used to inhibit growth of Gram positive
bacteria.
 Colistin is used to inhibit growth of Gram negative
bacteria.
 Nystatin is used to inhibit growth of fungal organisms.
 They are used for control of bacterial contaminants in tissue
cultures used in isolation of viruses e.g. Penicillin etc.
 They are used in more than one antibiotic (battery
antibiotics) to show the pattern of drug sensitivity of an
organism and constitutes as a simple method of timing (used
in form of drug sensitivity testing of an organism).

Antimicrobial (drug) resistance.

Antimicrobial resistance which is making difficulty in treatment of
some infectious diseases is due to:
 Extensive use and misuse of antimicrobial drugs which
favours emergence and survival of resistant strains of micro-
organisms such as Staphylococci, gonococci, Meningococci,
pneumococci Enterococci and other Gram negative bacteria
e.g. Salmonella, Shigella, Kle3bsiella, Pseudomonas and
Myco. tuberculosis.
 5
 Bacteria becomes resistant to antimicrobial agents through
the following mechanisms:
Production of enzymes which inactivate and modify
antibiotics.
Cause changes in the bacterial cell membrane thus
preventing the up take of an antimicrobial.
Modification of the target so that it no longer interacts
with the antimicrobial.
Development of metabolic pathways by the bacteria so
as to bypass the sites of antimicrobial action.
Mutation of the bacteria:
This arises during cell division (binary fusion) or
spontaneous cell division.
This occurs in two ways:
 Stepwise mutation: This involves a series of small steps of
mutation resulting into high levels of resistance.
 One step mutation: Resistance develops suddenly with the
first exposure with the drugs.
Transformation: This involves the transfer of genetic
materials which might be carrying resistance genes
through the agency of DNA.
Transduction: The plasmid determining penicillin
resistance in Streptococcus is transferred from cell to cell.
This is involved in the DNA. The drug resistance may be
mediated by R-factor.

Specimen inoculation.
Broth inoculation
 Sterilize a straight wire-loop with a Bunsen burner flame.
 Open the broth bottle and sterilize its mouth with a flame.
 Using a sterile wire-loop, pick a portion of the specimen and
mix it with the broth.
 Decontaminate the wire-loop using a Bunsen burner flame.
 Incubate the culture broth at the right temperature,
atmospheric conditions and adequate period of time.

Media in petri-dishes inoculation

This method is referred to as plating out or looping out method. It
is designed to give single colonies of the growing organism. The
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organism must be isolated in pure culture before it can be
identified and tested for drug sensitivity testing.
Steps.
 Dry the surface of the culture plates by placing the plate in
an incubator at 370C for 30-40 minutes.
Nb. Moisture will interfere with colonial morphology of the
growing organisms.

Plate streak-method.
Procedure
 Using a sterile wire-loop, make a smear of a loopful of
specimen or Bacterial cells over the surface area of the
agar plate in a continuous dilution, so that the cells are
separated from each other as shown below.

 Incubate the plate at 370C for 18-24 hrs.

 Observe the growth of the micro-organisms.
Precautions
 Surface of the culture medium should be dry because
moisture in the medium may lead to the spread of the
organism.

ANTIMICROBIAL SENSITIVITY TESTING.

This is done in the treatment and control of infectious diseases
caused by pathogenic organisms that are often resistant to drugs.
Sensitivity (susceptibility) testing is used to select effective
antimicrobial drugs.
The purposes of sensitivity testing are as follows:
 To guide the clinician to select the right drug to be given to
an individual patient.
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 For epidemiological purposes so that the drug resistance
pattern in a given community can be reflected for public
health importance.
This can be performed using:
A disc diffusion technique.
A dilution technique.

DISC DIFFUSION TECHNIQUE.

This is the commonly used method in the laboratories. There are
two methods.
Stokes method.
Kirby-Bauer method.

1. STOKES METHOD.
This is a diffusion method in which test organisms and control
organisms are inoculated in the same Agar plate.
Procedure
 Take a sensitivity testing agar plate and divide it into two
halves.
 Inoculate one half with the test organisms and the other half
with standard (control0 organisms.

 Place a disc of one antibiotic or multi-drug disc of antibiotics

2cm from the side of the plate and on the gulf between the
control organisms and the test organisms.
 Place another drug disc on the other side of the plate.
 Incubate at 370C for 18-24 hrs.
 Compare the zones of inhibition of the test organisms with
that of the control organism.
Results
A zone of inhibition comparable or greater than that of the
control organism (is reported as ‘sensitive (S).
 8
This means that the drug can kill the organism in vivo (in the
body) when given in the right dosage.
A small zone of inhibition around the test organism is
reported as ‘intermediate (I) or ‘slightly sensitive (SS).
This means that the drug can kill the organism in vivo (in the
body) when given at high dosage. It can also result into
resistance.
No zone of inhibition around the test organism is reported as
‘resistant (R).
This means that the drug cannot kill the organism in vivo (in
the body) irrespective of the dosage.

2. KIRBY-BAUER METHOD.
This is a method that directly measures antimicrobial activity.
Bacterial isolate in question is incubated in the same in-vitro
environment with the antimicrobial agent(s) in question to
determine the impact of a drug’s presence on bacterial growth.
Principle:
A known concentration of an antibiotic is placed on a filter paper
disk. The disk is placed on the surface of an agar plate that has
been inoculated with a bacterial culture. The antibiotic diffuses
out into the medium. The susceptibility of the organism to that
antibiotic is indicated by a clear zone of inhibition around the disk
so as to provide a qualitative result.

Method:
 Prepare bacterial suspension in which turbidity of the
organism inoculums is adjusted to density equal to 0.5
McFarland standards.
 Swab the organism onto the surface of Mueller-Hinton agar
(MHA) plate in order to make uniform distribution of the test
organism and let it stand for 15 minutes.
 Place antibiotics disc (single disc or multi-disc) on the
surface of the MHA plate.
 Incubate at 37C in ambient air for 16 to 18 hours.
 Examine growth and measure zone of inhibition’s diameter
around each antibiotic disk using reflective light using a
McFarland 0.5 table.
 9
 Place plates a few inches above a black, non reflecting
surface, measure from back of plate (agar side).The
diameter of the zone of inhibition determines the amount of
the drug in the disk that is susceptible to the test organism.
 The test must be vigorously standardized since inhibition
zone size is also dependent on the inoculums size,
composition of the media, incubation conditions, moisture,
and potency of antibiotic and thickness of the agar.

Reading and interpretation of sensitivity test

After (18-24) hrs of incubation, the plates are examined and
diameters of zone of inhibition measured to the nearest whole
millimeter by use of a caliper ruler. The diameter of the zone of
inhibition for individual antimicrobial agent is translated into
Susceptible or Sensitive (“S”), Intermediate (“I”), and Resistant
(“R”) categories by referring to a standard interpretative table
(McFarland 0.5 table).

29 mm

Results
A zone of inhibition comparable or greater than that of the
control organism (is reported as ‘sensitive (S).
This means that the drug can kill the organism in vivo (in the
body) when given in the right dosage.
A small zone of inhibition around the test organism is
reported as ‘intermediate (I) or ‘slightly sensitive (SS).
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This means that the drug can kill the organism in vivo (in the
body) when given at high dosage. It can also result into
resistance.
No zone of inhibition around the test organism is reported as
‘resistant (R).
This means that the drug cannot kill the organism in vivo (in
the body) irrespective of the dosage.

Factors affecting disc sensitivity testing.

1) Nature of the media:
Media should be free from inhibitory substances such as
antimicrobial agent (antibiotics), reducing substances etc.
2) Depth of the Agar:
The depth of the Agar should be 4mm or a volume of 15-20mls of
molten agar.
Thin agar media will lead to diffusion of the drug into the medium
hence giving high zone of inhibition.
3) Dryness of the media: Culture media should be free from
moisture.
 Moisture may lead to the spread of the organism and dilution
of the drug.
 Dry media will not allow diffusion of the drug into the media
hence false negative results.
4) Incubation condition:
 Time: Incubation period is 18-24 hrs except Myco.
tuberculosis which requires up to 8weeks.
 Temperature: Most organisms require temperatures of 35-
37C.
 Low temperatures give low growth of organism hence large
zone of inhibition giving false positive results.
 High temperatures will lead to no growth of organism hence
large zone of inhibition.
5) Size of the inoculums:
Size of the inoculums should be adjusted to produce numerous
small colonies but not a continuous film of organisms.
 Heavy inoculums produces small zone of inhibition hence
giving false negative results.
 Light inoculums produces large zone of inhibition hence
giving false positive results.
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6) pH = 7.2 to 7.4
Some organisms will not grow in acidic and alkaline pH.
7) Concentration of the sensitivity disc:
 High concentrations give large zone of inhibition while low
concentrations give small zone of inhibition.
8) Concentration of agar should be 1-2 %.
9) Previous exposure to drugs.
10) Proper storage:
 Store at -20C for 1year.
 Store at 2-8C for a short period in a desiccators.
This is to preserve the potency of the sensitivity disc.

Summary on drug susceptibility testing

1. Gram positive drug disc.
These are drugs that inhibit bacterial cell wall peptidoglycan
synthesis.
Drug disc KGL ¼
All penicillins e. g.
Benzyl penicillin
Amphicillin
Amocycillin
Flucloxacillin
Cloxacillin
Carbenicillin
Vancomycin
Erythromycin
Methicillin
Lincomycin
Chloramphenicol
Minocyclin
Cotrimoxazole.
2. Gram negative drug disc.
These are drugs that:
 Inhibit bacterial cell wall peptidoglycan synthesis.
 Inhibit Nucleic acid synthesis.
Drug disc KGL 2/5
Cefuroxime
Cephradine
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Cefotaxime
Tetracycline
Doxycline
Nalidixic acid
Norfloxacin
Ceprofloxacin
Augmentin
Gentamycin
Chloramphenicol
Amphicillin
Cotrimoxazole.
3. Both Gram negative and Gram positive drug disc.
These are drugs that:
 Inhibit bacterial protein synthesis.
 Inhibit Nucleic acid synthesis.
Drug disc KGL 2/5
Cefuroxime
Erythromycin
Tobramycin
Tetracycline
Doxycline
Nalidixic acid
Norfloxacin
Kenamycin
Augmentin
Gentamycin
Chloramphenicol
Amphicillin

II. Overview
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A. Goal of antimicrobial susceptibility testing is to determine by
in vitro methods the extent of an organism’s acquired
resistance and predict the in vivo response of a bacterial
isolate against an antimicrobial agent
1. Gene mutations and acquisition of new genetic material (i.e.,
plasmids) cause differences

2. Causes antimicrobial susceptibility differences between species

B. Standardization of testing
1. Optimize bacterial growth conditions so any inhibition of growth
is due to antimicrobial agent

2. Optimize conditions for maintaining antimicrobial integrity and

activity so any inhibition of bacterial growth can be attributed to
organism resistance mechanisms

3. Maintain reproducibility and consistency of results

4. All test components and conditions – agar; antimicrobial

formulations and concentrations; organism suspension
concentration; incubation temperature, atmosphere and time
C. Bacterial inoculums purity and size - direct colony
suspension method (commonly used method)
1. Touch 4-5 colonies of same morphology with sterile loop or
swab, culture is 18-24 hours old from nonselective (e.g. sheep
blood agar) agar and suspend in 0.85% sterile saline

2. Adjust organism suspension turbidity to match a 0.5 McFarland

density standard (equivalent to 1.5 X 108 CFU) using:

a. Densitometer

b. Unaided eye – view suspensions against black-lined

background
D. Growth medium – Mueller-Hinton (M-H) base
1.
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2+ 2+
2. Cation concentration: 50-100 μg/L Ca and 20-35 μg/L Mg
(affects the susceptibility results when certain combinations of
bacterial species and antibiotic are tested)

3. Agar depth =

4. If necessary for organism growth, add blood and serum

supplements to M-H base
E.Incubation atmosphere, temperature and duration
1. Ambient air, 35C, 16 -18 hours

2. If organism doesn’t grow in these conditions, must utilize

specialized interpretation criteria
 15
F. Antimicrobial-impregnated disks – standard concentration
1. Store antimicrobial-impregnated disks at -20C or lower in an
anhydrous condition
2. Maintain a small working supply at refrigerator temperatures in
a desiccator
3. Allow disks to come to room temperature before opening
dessiccator or using
G. Quality Control - test standard reference bacterial strains that
have a defined antimicrobial susceptibility to the drugs tested
1. Store standard bacterial strains in a condition that minimizes
the possibility of mutation
a. Long-term storage - freeze below -20C or preferably
below -60C after suspension in a stabilizer such as
defibrinated whole blood, 50% fetal-calf serum in
bacteriologic broth or 10% glycerol in broth for long term
storage
b. Short-term storage – 2-8C on nutrient agar slant for 2
weeks
c. Obtain a new stock culture when aberrant results
obtained
d. Utilize a fresh subculture when control strain is used
2. Frequency of running quality control – per manufacturer or
regulatory agency
H. Interpretation of results – utilize standard criteria to
determine if organism is susceptible, intermediate or
resistant to a certain antimicrobial agent
1. Susceptible: antimicrobial agent should be affective in vivo
 16
2. Intermediate: for these isolates MICs approach usually
attainable levels and response rates may be lower than for
susceptible isolates. Implies clinical applicability in body sites
where the drugs are physiologically concentrated (i.e., beta-
lactams in urine) or when high dosage of the drug can be used
(i.e., beta-lactams). Also indicates a “buffer zone, which
should prevent small, uncontrolled technical factors from
causing major discrepancies in interpretation.
3. Resistant: antibiotic probably will not be effective in vivo, this
category provides the best correlation between in vitro and in
vivo results.
III. Testing Methods
A. Methods that directly measure antimicrobial activity –
bacterial isolate in question is incubated in the same in vitro
environment with the antimicrobial agent(s) in question to
determine the impact of a drug’s presence on bacterial
growth
 17

B. Methods that directly detect specific resistance mechanisms

1. Beta-lactamase production: enzymes produced by bacteria that
break down beta-lactam antibiotics rendering the antibiotic
ineffective

a. Chromogenic Cephalosporinase Test (Cefinase test)

Methodology

b. Chromogenic (yellow) cephalosporins nitrocefin will turn

red in the presence of beta-lactamase and hydrolysis of
its beta-lactam ring

c. Uses (see package insert)

i. Haemophilus influenzae resistance to ampicillin
ii. S. aureus resistance to penicillin
iii. Neisseria gonorrhoeae & Neisseria meningitides
resistance to penicillin
iv. Anaerobic bacteria resistance to penicillin for
identification purposed
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2. Detection of PBP 2a – rapid latex agglutination assay for
detection of modified penicillin binding protein found in
Methicillin/Oxacillin Resistant Staphylococcus aureus (encoded
for by mecA gene)
C. Screening/Supplemental Testing Methods
1. Detection of Methicillin/Oxacillin-resistant Staphylococcus
aureus (MRSA) – Kirby Bauer susceptibility using cefoxitin disk
(this method will not detect MRSA that is mecA-mediated)
a. Prepare inoculum using direct colony suspension
method and adjust to 0.5 McFarland standard
b. Set up Kirby-Bauer susceptibility with cefoxitin disk
(follow standard protocol)
c. Incubate at 33-35C (maximum = 35C) for 24 hours
d. Measure zone of inhibition with reflected light
e. Interpretation = > 22 mm = oxacillin susceptible
< 21 mm = oxacillin resistant
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f. If MRSA, then all beta-lactam antibiotics are resistant in
vivo regardless of in vitro testing (due to the altered
binding protein PBP2a)
D. Enterococcus sp. (Enterococcus faecalis, Enterococcus
faecium)
For isolates from sterile body sites or if causing a systemic,
life-threatening infection, a cell-wall active agent (penicillin or
ampicillin; then vancomycin) plus an aminoglycoside
(gentamicin or streptomycin) are used for treatment to
achieve synergistic effect

Intrinsically “R” to cephalosporins (due to low affinity

binding proteins), oxacillin and clindamycin (may test “S” in
vitro)
1. Synergy Screen (to determine if aminoglycoside will act
synergistically with “S” cell wall antibiotic)
2. Test Gentamicin 500 ug/ml and streptomycin 1000 ug/ml
 20
3. Interpretation:
If the Enterococcus sp. is sensitive to the high level concentration
of gentamicin or
streptomycin, then that aminoglycoside should act synergistically
with a cell wall
antibiotic (i.e., penicillin) if the organism is “S” to that cell wall
antibiotic

Example:
Results:
Penicillin (S) Streptomycin Synergy Screen (S) Gentamicin
Synergy Screen (R)
Interpretation:
Penicillin should act synergistically with streptomycin (but not
gentamicin)

E. Detection of Vancomycin resistant Enterococcus (VRE) –

vancomycin screen (incubate full 24 hours)
1. Most common VRE is Enterococcus faecium
2. Confirm vancomycin resistance via different test methodology
F. Extended-spectrum beta-lactamases (ESBLs) – mediate
resistance to extended-spectrum cephalosporins (i.e.,
cefotaxime, ceftriaxone & ceftazidime), monobactams (i.e.,
aztreonam) and extended-spectrum penicillins (i.e., ticarcillin,
piperacillin)

1. Beta-lactamase inhibitors (i.e., clavulanate, sulbactam, tazobactam)

block the activity of ESBLs

2. ESBLs have no effect on cephamycins (i.e., cefoxitin) or

carbapenems (i.e., imipenem or meropenem)

3. Found in gram-negative organisms, especially E. coli, Klebsiella

pneumoniae & K. oxytoca, Proteus mirabilis (not limited to these
organisms), standardized testing only for these organisms
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4. Screen for ESBLs on all E. coli and Klebsiella sp. isolates (except for
UTIs)

a. Example: disk diffusion: Aztreonam zone < 27 mm =

possible ESBL producer

b. Example: MIC: Aztreonam = > 2ug/ml is possible ESBL

producer

c. Other clues for ESBL production:

i. aztreonam &/or ceftazidime “I” or “R,” yet other beta-
lactams are “S”
ii. normal patterns of resistance to beta-lactam antibiotics not
followed
iii. If patterns in (d) or (e) demonstrated (difficult to detect),
perform ESBL confirmatory testing (disk diffusion or MIC)
cefotaxime (R) with cefotaxime/clavulanic acid (S) &/or
ceftazidime (R) with ceftazidime/clavulanic acid (S) then
confirmed ESBL producer
Report all cephalosporins, penicillins and
aztreonam as (R) regardless of in vitro test results
Report cephamycins and carbapenems as tested
Add a comment to the report “Confirmatory tests of this
isolate indicate unusual resistance (ESBL production)”
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G. Streptococcus pneumoniae – Screening for Penicillin
Resistance
1. Penicillin Screen: Perform a Kirby-Bauer susceptibility using
Mueller Hinton Agar supplemented with 5% sheep blood and
oxacillin disk
2. Interpretation:
zone > 20 mm = penicillin susceptible and also susceptible to
ampicillin, amoxicillin, cefaclor, cefotaxime, ceftizoxime,
ceftriaxone, cefuroxime, imipenem (list not all inclusive)

zone < 19 mm presumptive for penicillin resistance: must be

confirmed by
MIC
 23

Correlation of Antimicrobial Susceptibility Pattern to Identification

H. Predictable Patterns of Susceptibility

Examples of “Predictable Patterns of Susceptibility”

Predictable Susceptibility*
Organism (If)
(Then)
“S” to ampicillin and beta-
Any organism “S” to ampicillin
lactamase inhibitor combination
Any GNR “S” to gentamicin “S” to amikacin
Any GNR “S” to first-generation “S” to second- and third-
cephalosporin generation cephalosporins
Any GNR “S” to second- “S” to third generation
generation cephalosporin cephalosporin
Enterobacteriaceae “S” to gentamicin
Klebsiella pneumoniae
“R” to ampicillin
Citrobacter koseri
Enterobacter sp., Morganella sp. “R: to ampicillin and first-
Citrobacter freundii generation cephalosporin
“R” to nitrofurantoin and
Proteus mirabilis
tetracycline
“R” to ampicillin and first-
generation cephalosporin
Serratia sp.
(“R” to tobramycin in vivo, but
tests “S” in vitro)
Any gram-positive bacteria
(except for Lactobacillus,
“S” to vancomycin
Pediococcus, Leuconostoc,
Erysipelothrix rhisiopathiae)
MRSA (methicillin/oxacillin-
resistant S. aureus also know as “R” to all beta-lactam antibiotics
multi-drug resistant S. aureus)
Beta-hemolytic streptococci “S” to penicillin
*If an organism deviates from this pattern, the id and/or the validity
of the susceptibility should be questioned

Other patterns to be aware of:

 24
E. coli, Proteus mirabilis, Klebsiella sp. (possibly other Enteric
organisms) if “R” to
ceftazidime &/or aztreonam & “S” to other cephalosporins (possible
extended-spectrum beta-lactamase producer) – perform confirmation
testing

S. aureus and Staphylococci coagulase negative “I” or “R” to

vancomycin – perform confirmation testing and inform appropriate
health care agencies.

Enterococci = “R” to vancomycin = VRE – perform confirmation

testing
I.Example of cumulative antibiogram
Clinical Microbiology Antibiogram
Antimicrobial Susceptibility of Common Organisms (6 months)
Reported as % Susceptible
Isolated
Antimicrobial Agent
Organism
Cefuroxime

Cefotaxime

Gentamicin
Ciprofloxac

Piperacillin
Aztreonam

Cephalothi
Sulbactam

Ceftazidim

Nitrofurant
Ampicillin/

Imipenem
Cefepime
Ampicillin

Cefazolin
Amikacin

Cefoxitin

Trimeth/
Isolates

e (3rd)
Total

(2nd)

Pip/
(4th)
in

Escheric 168 10 5 9 7 7 8 9 9 9 7 9 8
80 82 99 99 93 99
hia coli 0 0 3 9 8 8 2 9 3 0 0 9 4
Klebsiell
a 10 9 6 6 8 9 10 9 10 4 5 9 8
280 0 61 80 95 96
pneumon 0 5 0 0 0 5 0 8 0 2 0 5 9
iae
Pseudo
monas 114 4 6 8 9
81 66 83 60 79
aerugino 2 5 8 0 0
sa

For urinary tract isolates only

 25
1. In this example
100 % of the Escherichia coli tested were susceptibile to
amikacin
53% of the Escherichia coli tested were susceptibile to
ampicillin
0% of the Klebsiella pneumoniae tested were susceptibile
to ampicillin

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Slide 10 Slide 9 Slide 8 Slide 7 Slide 6 Slide 5
26
Slide 16 Slide 15 Slide 14 Slide 13 Slide 12 Slide 11
27
 28

Slide 17
Slide 18

Example: beta-lactamases, group of

Slide 19

enzymes that hydrolyze (blow-up) the beta-

lactam ring, once the ring is broken, the
antibiotic is useless
Slide 20
Slide 21
Slide 22
Slide 28 Slide 27 Slide 26 Slide 25 Slide 24 Slide 23
29
Slide 34 Slide 33 Slide 32 Slide 31 Slide 30 Slide 29

Continue to page 2 of notes

30
 31

Slide 35
Slide 36
Slide 37
Slide 38
Slide 39

Learning Objective Study Questions – Key

1. When performing susceptibility testing, which is a standardized

method of inoculum preparation?
From an 18-14 hour, nonselective agar plate:
a. touch one bacterial colony and emulsify in 0.85% saline
until the turbidity is equivalent to a 0.5 McFarland
density standard
 32
b. touch one bacterial colony and emulsify in a broth and
incubate at 35C for 8 hours
c. touch 4 to 5 colonies of similar morphology and emulsify in
0.85% saline until the turbidity is equivalent to a 0.5
McFarland density standard
d. swipe an area in the primary streak area to obtain a
heterogeneous mixture of all bacterial types and emulsify in
a broth until observable turbidity is detected

2. Except for organisms with specific growth requirements,

antimicrobial susceptibility tests are incubated in:
a. Ambient air at 35C
b. Ambient air at 37C
c. 5-7% CO2 at 35C
d. 5-7% CO2 at 37C
e. Ambient air or 5-7% CO2 at a temperature range of 35C -
38C

3. For a disk diffusion test, the Mueller-Hinton agar needs to be at

a depth of _________ with a pH of ________.
a. 4 mm; 7.2-7.4
b. 4 cm; 7.2-7.4
c. 7 mm; 4.2-4.4
d. 7 cm; pH 4.2-4.4

4. A microdilution MIC of ampicillin vs. E. coli was interpreted as

follows:

Growth control well = growth

Sterility control well = no growth
Ampicillin 32 ug/ml = no growth
Ampicillin 16 ug/ml = no growth
Ampicillin 8 ug/ml = growth
Ampicillin 4 ug/ml = growth

The MIC should be reported as:

a. 4 ug/ml
b. 8 ug/ml
c. 16 ug/ml
 33
d. 32 ug/ml
e. Unable to interpret due to invalid control results

5. Beta-lactamases are a group of enzymes:

a. That are a chemical part of beta-lactam antibiotics allowing
them to inhibit cell wall synthesis
b. That are a chemical part of beta-lactam antibiotics allowing
them to bind to a target protein in bacterial cell walls
c. Produced by bacteria and act to hydrolyze the beta-
lactam ring within beta-lactam antibiotics causing the
antibiotic to be ineffective
d. Produced by bacteria and act to hydrolyze the beta-lactam
ring within an organism’s cell wall disrupting cell wall
synthesis
 34

Laboratory Assignment – Performing Kirby-Bauer Susceptibilities

Name: Date:

Possible points:

Assignment Objectives:
Upon completion of this laboratory exercise, you will gain
awareness and the ability to:
1. Perform a Kirby-Bauer diffusion susceptibility test.
2. Interpret Kirby-Bauer susceptibility test results, including the
use of standardized interpretative criteria.
3. Evaluate the acceptability of Kirby-Bauer susceptibility test
results based on data of an organism’s characteristic
antimicrobial profile.

Part I Day 1 - Perform Kirby-Bauer Antimicrobial Susceptibility (on

one single organism)
1. Using dacron-tipped swab or a bacteriologic loop, touch the
tops of 4 to 5 bacterial colonies of the same morphology.
2. Transfer the bacteria to a tube containing saline.
3. Match the density of the bacterial suspension to the 0.5
McFarland density standard by visual inspection (look at lines
on card through both suspensions)
4. Within 15 minutes of adjusting the bacterial suspension, insert a
cotton-tipped swab into the suspension, rotate the swab firmly
against the wall of the tube to express excess fluid, and streak
the surface of a Mueller-Hinton agar plate.
5. Swab the surface of the plate three times, rotating the plate
approximately 60 each time (i.e., make a lawn of organism)
6. Allow 3 to 5 minutes, but no more than 15 minutes for the
inoculum to dry. Dispense the antibiotic impregnated disks on
the surface of the inoculated agar.
 35
7. Tamp the disks down firmly onto the surface of the agar. Do
NOT move the disks once they have touched the agar surface.
8. Incubate agar side up for 18-24 hours in a non-CO2 incubator
 36
Part II Day 2– Interpret Kirby-Bauer Antimicrobial Susceptibility

1. Measure in mm the diameter of zones of inhibition

Measure from completely clear area to completely
clear area across the disk using the appropriate light
(reflective or transmitted) based on the
organism/antibiotic combinations.

29 mm

2. Using the provided ‘Zone Diameter Interpretive Standards

Criteria Chart’, record the sensitivity results below.

Diameter in Interpretation
Organism Name Antibiotic
mm (S, I, R)

3. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
 37
Example of Kirby Bauer Zone Diameter Interpretive Standards
Criteria
 38

Example of Kirby Bauer Zone Diameter Interpretive Standards

Criteria (cont. page 2)
39
 40

Example Clinical Microbiology Antibiogram

 Clinical Microbiology Antibiogram
41
Antimicrobial Susceptibility of Common Organisms (6 months)
Reported as % Susceptible
Isolated Antimicrobial Agent
Organism

tazobactam
Cefuroxime

Cefotaxime

Gentamicin
Ciprofloxac

Norfloxacin

Piperacillin
Aztreonam

Cephalothi

Ceftazidim

Nitrofurant

Mezlocillin
Ampicillin/

Imipenem
Cefepime
Ampicillin

Cefazolin
Amikacin

Cefoxitin

Trimeth/
Isolates

Sulbac

e (3rd)
n (1st)

Sulfa
Total

oin
(2nd)
(2nd)

(3rd)

Pip/
(4th)
(1st)

in


Gram-negative Bacteria
Acinetobacter sp. 86 10 15 35 0 0 25 25 26 26 30 45 55 95 45 15 15 45 50
0
Citrobacter koseri 90 10 0 60 10 90 90 98 98 10 10 10 10 10 10 90 10 85 85 10 98
0 0 0 0 0 0 0 0 0 0
Citrobacter 18 10 0 35 93 15 15 20 20 87 85 99 81 98 10 86 81 75 75 81 53
freundii 2 0 0
Enterobacter 10 10 0 3 96 0 0 21 21 82 82 10 96 10 10 15 96 96 96 10 76
aerogenes 8 0 0 0 0 0
Enterobacter 47 10 0 4 83 0 0 18 18 78 78 97 90 98 99 23 90 67 67 80 83
cloacae 2 0
Escherichia coli 16 10 53 80 99 78 78 82 82 99 99 99 93 93 99 90 93 70 70 99 84
80 0
Haemophilus 16 56 96 10 91
1
influenzae 8 0
Klebsiella oxytoca 80 10 0 75 95 50 50 95 95 10 10 10 95 97 10 43 95 25 25 97 95
0 0 0 0 0
Klebsiella 28 10 0 61 95 60 60 80 80 95 95 10 96 98 10 42 96 50 50 95 89
pneumoniae 0 0 0 0
Morganella 46 10 0 4 91 0 0 43 43 91 91 10 52 96 91 60 5 43 43 52 61
morganii 0 0
 42

1
Cefuroxime = 98%, Clarithromycin = 100%
For urinary tract isolates only

For Student Use Only – this does not represent actual data
Example Clinical Microbiology Antibiogram

Clinical Microbiology Antibiogram

Antimicrobial Susceptibility of Common Organisms (6 months)
Reported as % Susceptible
Isolated Antimicrobial Agent
Organism
Cefuroxime

Ceftriaxone

Erythromyc
Cefotaxime

Gentamicin
Ciprofloxax

Norfloxacin
Clindamyci

Vancomyci
Tetracyclin
Sulbactam

Nitrofurant
Ampicillin/
Ampicillin

Penicillin
Oxacillin

Trimeth/
Isolates

(2nd)

Sulfa
Total

oin 
(3rd)
(3rd)
in

in


n

n
Gram-positive Bacteria
 43

Enterococcus 67 53
99 1 802 95 51 99 411 99
faecalis. 5
Enterococcus 45 34
56 1 732 61 32 56 601 70
faecium 1
Staphylococcus 14 10 10 10
95 76 94 8 98 99
aureus 20 0 0 0
Staphylococcus 26 10
42 8 92 96 0 0 88 99
aureus (MRSA) 9 0
Staphylococus sp. 12 10
66 30 61 99 28 7 85 65
coagulase neg 98 0
Streptococcus 22 10 10
86 89 73 57 81 60
pneumoniae 2 0 0
Streptococcus sp. 10
44 93 91 64 67 68
viridans grp. 0

Urinary tract isolates only
1
Urinary tract isolates only
2
Tested for high level susceptibility (500 ug/ml) to predict synergism

For Student Use Only – this does not represent actual data
 44

If actual K-B susceptibilities are not performed, these

pictures can be printed on transparent paper, cut out and
placed in a petri dish.

Pseudomonas aeruginosa IMP = imipenen, AZT =

aztreonam, CAZ = ceftazidime

Staphylococcus epidermidis P = penicillin, CC =

clindamycin, OX = oxacillin
45
 46

Laboratory Assignment – Beta-lactamase Testing and

Interpretation

Name: Date:

Possible points:

Assignment Objectives:
Upon completion of this laboratory exercise, you will gain
awareness and the ability to:
1. Perform a beta-lactamase test.
2. Explain the principle of the Cefinase beta-lactamase test.
3. Interpret beta-lactamase susceptibility test results.

Beta-lactamase testing:
1. The quality control results for the current lot of Cefinase
disks are shown below. The reactions were noted after 60
seconds of incubation.
Negative control organism = Haemophilus influenzae
ATCC 10211
Positive control organism = Staphylococcus aureus
ATCC 25913

Are these control results acceptable? Explain.

 47

2. The results for a Staphylococcus aureus are shown

below. This reaction was noted after 60 seconds of
incubation.

Interpret these results.

What do these results indicate?

 48

Laboratory Assignment – Microdilution Minimum Inhibitory

Concentration (MIC) Susceptibility Interpretation &
Evaluation

Name: Date:

Possible points:

Assignment Objectives:
Upon completion of this laboratory exercise, you will gain
awareness and the ability to:

1. Interpret MIC susceptibility test results, including the use

of standardized interpretative criteria.

2. Evaluate the acceptability of MIC susceptibility test results

based on data of an organism’s characteristic
antimicrobial profile.

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC
Interpretive Standards Criteria’ interpret the result as
susceptible, intermediate or resistant.
Organism tested:
Interpretation
Antibiotic MIC Result (susceptible, intermediate,
Tested resistant)
 49

3. Using the provided ‘Clinical Microbiology Antibiogram’

and known predictable patterns of susceptibility
information, do these results follow characteristic
antimicrobial susceptibility patterns? Explain.
50
51
52
 53

Example MIC Interpretative Criteria Page 1

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Staphylococcus sp.
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Beta-lactams – Penicillins
Penicillin-susceptible staphylococci are also susceptible to other
penicillins, beta-lactamase inhibitor combinations cephems, and
carapenems approved for staphylococcal infections. Peniclllin-
resistant, oxacillin-susceptible strains are resistant to beta-lactamase-
labile penicillins, beta-lactamase inhibitor combinations, relevant
cephems, and carbapenems. Oxacillin-resistant staphylococci are
resistant to all currently available beta-lactam antibiotics. Thus,
susceptibility or resistance to a wide variety of beta-lactam antibiotics
can be deduced from testing only penicillin and oxacillin.
<0.12
See note 4
Penicillin - >0.25
Oxacillin <2 - >4 For S. aureus
<0.25 - >0.5 For coag-neg
staphylococci
See Note 5
Beta-lactams – Penicillins (cont.)
Methicillin or <8 - >16 S. aureus only
Nafcillin <2 - >4

Carbenicillin <16 32 >64

Beta-lactams - Cephems (including cephalosporins I, II, III, and
IV)
Cefazolin <8 16 >32
Cephalothin <8 16 >32
Carbapenems
Imipenem <4 8-16 >32
 54

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Staphylococcus sp.
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Meropenem <4 8-16 >32
Glycopeptides
Vancomycin <4 8-16 >32 Rare
vancomycin-
resistant
staphylococci
have been
isolated. Send
any staphylococci
determined to
have an elevated
MIC for
vancomycin (>4
ug/ml) to a
reference lab.
Teicoplanin <4 8-16 >32
Aminoglycosides
Gentamicin <4 8 >16
Amikacin <16 32 >64
Kanamycin <16 32 >64
Netilmicin <8 16 >32
Tobramycin <4 8 >16
 55

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Staphylococcus sp.
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Macrolides
Azithromycin or <2 4 >8 Not routinely
<2 4 >8 tested and
Clarithromycin or <0.5 1-4 >8 reported against
Erythromycin organisms
isolated from the
urinary tract.
Tetracyclines
Tetracycline is the representative for all tetracyclines and the
results can be applied to doxycycline and minocycline. Certain
organisms may be more susceptible to doxycycline and minocycline
than to tetracycline.
Tetracycline <4 8 >16
Nitrofurantoins
Nitrofurantoin <32 64 >128 Urine only
Ansamycins
Rifampin <1 2 >4 Rifampin should
not be used alone
for
chemotherapy.
 56

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Staphylococcus sp.
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Fluoroquinolones
Staphylococcus sp. may develop resistance during prolonged therapy
with quinolones. Therefore, isolates that are initially susceptible may
become resistant within 3 to 4 days after initiation of therapy. Testing
of repeat isolates may be warranted.
Ciprofloxacin or <1 2 >4
Levofloxacin <2 4 >8
or <2 4 >8
Ofloxacin
Lomefloxacin or <2 4 >8 Urine only
Norfloxacin <4 8 >16
or <2 4 >8
Ofloxacin
Lincosamides
Clindamycin <0.5 1-2 >4 Not routinely
tested and
reported against
organisms
isolated from the
urinary tract.
Folate Pathway Inhibitors
Trimethoprim/ <2/38 - >4/76
sulfamethoxazole
 57

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Staphylococcus sp.
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Sulfonamides <256 - >512 Urine only
Sulfisoxazole can
be used to
represent any of
the currently
available
sulfonamide
preparations.
Trimethoprim <8 - >16 Urine only
Phenicols
Chloramphenicol <8 16 >32 Not routinely
tested against
organisms
isolated from the
urinary tract.

Notes:
1. Historically, resistance to the antistaphylococcal beta-
lactamase-stable penicillins has been referred to as
“methicillin resistance.” Even though MRSA (methincillin-
resistant S. aureus) or MRS (methicillin-resistant
staphylococci) is still commonly used to denote such
resistance, methicillin is no longer the agent of choice for
testing.

2. For oxacillin-resistant Staphylococcus aureus (MRSA) and

coagulase-negative staphylococci,
all penicillins, cephems, and other beta-lactams such as
amoxicillin/clavulanic acid, ampicillin.sulbactam,
ticarcillin/clavulanic acid, piperacillin/tazobactam, and
 58

imipenem may apppear active in vitro, but are not effective

clinically and isolates should NOT be reported as
susceptible. This is because most cases of documented
MRSA & Methicillin-resistant staphylococci (MRS) infections
have responded poorly to beta-lactam therapy.
Notes (cont.)

3. Routine testing of S. saprophyticus isolated from urine

is not advised because infections respond to concentrations
achieved in urine of antimicrobial agents commonly used to
treat acute, uncomplicated urinary tract infections (i.e.,
nitrofurantoin, trimpthoprim/sulfamethoxazole, or a
fluoroquinolone)

4. Resistant strains of S. aureus produce beta-lactamase.

Penicillin is the antibiotic of choice for such detection. A
penicillin MIC of <0.03 ug/ml implies lack of beta-lactamase
production. Staphylococci with MICs of 0.06-0.12 ug/ml may
or may not produce beta-lactamse. An induced beta-
lactamase test can clarify these MICs. A positive beta-
lactamase test implies resistance to penicillin, ampicillin,
amoxicillin, carbenicillin, ticarcillin, mezlocillin, and
piperacillin.

5. Of the antistaphylococcal, beta-lactamase-stable

penicillins, oxacillin may be tested and results can be applied
to other penicillinase-stable penicillins, cloxacillin and
dicloxacillin. Oxacillin is preferred as it detects more
heteroresistant strains. Most MRS are usually resistant to
multiple antibiotics, including all other beta-lactams,
aminoglycosides, macrolides, clindamycin, and tetracycline.
The observation of multiple resistance should be a clue to
the possibility of oxacillin resistance.
 59

Clinical Checklist
Following demonstration by the instructor and using the
maintenance manual and instrument manual(s), the
student will be able to:
Mastery Observed Discuss
(works with demonstration procedure
some but did
independence) not
observe
Kirby-Bauer
Susceptibility
Test
Microdilution
Susceptibility
Test
Beta-
lactamase
Test

Student
Name_________________________________________
_____
 60

Total number of (lab procedure) performed

________________________________
Name of Clinical Supervisor/ Bench
Instructor______________________________
Signature of Clinical Supervisor/ Bench
Instructor___________________________
Date:__________________________________________
____________
 61

Example Exam Questions- Key

1. An oxacillin microdilution MIC was performed on

Staphylococcus aureus with the
following results

Gro Steri
16 8 4 2 1 0.5 0.25
wth lity
ug/m ug/m ug/ ug/m ug/m ug/m ug/m
Cont Cont
l l ml l l l l
rol rol

The MIC is reported as:

a. 16.0 ug/ml
b. 2.0 ug/ml
c. 1.0 ug/ml
d. Test is invalid based on control results

2. An ampicillin microdilution MIC was performed on

Escherichia coli with the following results:

Gro Steri
16 8 4 2 1 0.5 0.25
wth lity
ug/m ug/m ug/ ug/m ug/m ug/m ug/m
Cont Cont
l l ml l l l l
rol rol

The MIC is reported as:

a. <0.25 ug/ml
b. >0.25 ug/ml
 62

c. <16 ug/ml
d. >16 ug/ml
e. Test is invalid based on control results

3. Kirby-Bauer and dilution (MIC) susceptibility testing is

performed with what medium?
a. Brain heart infusion
b. Mueller-Hinton
c. Sheep blood
d. Trypticase soy

4. What are the best incubation requirements when

performing a Kirby Bauer
susceptibility test to detect methicillin resistant
Staphylococcus aureus (MRSA)?
a. 30°C for 16-18 hours in ambient air incubator
b. 30°C for 16-18 hours in CO2 incubator
c. 35°C for 16-18 hours in ambient air incubator
d. 35°C for 24 hours in ambient air incubator
e. 35°C for 24 hours in CO2 incubator

5. In a disk diffusion susceptibility test, which of the following

can result if disks are placed on the inoculated media and
left at room temperature for an hour before incubation?
a. The antibiotic would not diffuse into the medium, resulting
in no zone of inhibition
b. Zones of smaller diameter would result
c. Zones of larger diameter would result
d. There would be no effect on the final zone diameter

6. A 21-year-old female diagnosed as having pneumonia

caused by Streptococcus
pneumoniae is not responding to penicillin therapy. Which
of the following tests
 63

should be performed on the isolate to determine this

organism’s susceptibility to
penicillin agents?
a. Beta-lactamase test
b. Oxacillin disk diffusion
c. Penicillin disk diffusion
d. Unable to perform susceptibility on Streptococcus
pneumoniae

7. A culture of Staphylococcus aureus is noted on a report

form to have the following Kirby-Bauer results:

Penicillin G: Resistant
Oxacillin: Susceptible
Vancomycin: Resistant

The next best step is to:

a. Confirm the penicillin G result

b. Confirm the vancomycin result
c. Perform a beta-lactamase test
d. Report the susceptibility without further action
 64

Example MIC Interpretative Criteria

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Enterobacteriaceae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Beta-lactams - Penicillins
<8 Class
Ampicillin 16 >32 representative for
ampicillin/amoxicil
lin
Mezlocillin or <16 32-64 >128
Piperacillin <16 32-64 >128
Ticarcillin <16 32-64 >128
Carbenicillin <16 32 >64
Beta-lactams/Beta lactamase Inhibitor Combinations
Amoxicillin/ <8/4 16/8 >32/16
clavulanic acid or <8/4 16/8 >32/16
<16/4 32/4- >128/4
Ampicillin/sulba <16/2 64/4 >128/2
ctam 32/2-
Piperacillin/ 64/2
tazobactam
Ticarcillin/
clavulanic acid
 65

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Enterobacteriaceae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Beta-lactams - Cephems (including cephalosporins I, II, III, and
IV) – (Parenteral)
For Salmonella sp. & Shigella sp., 1st and 2nd generation
cephalosporins may appear active in vitro, but are
not effective clinically and should not be reported as susceptible.
Strains of Klebsiella sp. and E. coli that produce ESBLs may be
clinically resistant to therapy with penicillins, cephalosporins, or
aztreonam, despite apparent in vitro susceptibility to some of these
agents. For all ESBL producing strains, the test interpretations
should be reported as resistant for all penicillins, cephalosporins, and
aztreonam. (See procedure for ESBL screening and confirmatory
tests.)
Enterobacter, Citrobacter, and Serratia sp. may develop resistance
during prolonged therapy with 3rd generation cephalosporins.
Therefore, isolates that are initially susceptibile may become resistant
within 3 to 4 days after initiation of therapy. Testing of repeat isolated
may be warranted.
Cefazolin <8 16 >32 Cephalothin can
Cephalothin <8 16 >32 be used to predict
activity of
cephalothin,
cephapirin,
cephadine,
cephalexin,
cefaclor and
cefadroxil.
Cefazolin,
cefuroxime,
cefpodoxime,
cefprozil, and
 66

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Enterobacteriaceae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
loracarbef
(urinary isolates
only) may be
tested individually
because some
isolates may be
susceptible to
these agents
when resistant to
cephalothin.
Cefamandole or <8 16 >32
Cefonicid or <8 16 >32
Cefuroxime <8 16 >32
sodium

(parenteral)
Cefepime <8 16 >32
Cefmetazole <16 32 >64
Cefoperazone <16 32 >64
Cefotetan <16 32 >64
Cefoxitin <8 16 >32

Cefotaxime or <8 16-32 >64 Cefotaxime or

Ceftizoxime or <8 16-32 >64 ceftriaxone
Ceftriaxone <8 16-32 >64 should be
reported on
isolates from
CSF.
Ceftazidime <8 16 >32
Moxalactam <8 16 >32
 67

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Enterobacteriaceae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Cephems (Oral)
Cefuroxime axetiel <4 8-16 >32
(oral)
Loracarbef <8 16 >32 Urine only
Cefaclor <8 16 >32
Cefixime <1 2 >4
Carbapenems
Imipenem or <4 8-16 >32
Meropenem <4 8-16 >32
Monobactams
Aztreonam <8 16 >32
Aminoglycosides
For Salmonella sp. & Shigella sp. aminoglycosides may appear
active in vitro but are not effective clinically and isolates should not be
reported as susceptible.
Gentamicin <4 8 >16
Amikacin <16 32 >64
Kanamycin <4 8 >16
Netilmicin <4 8 >16
Tobramycin <4 8 >16
Tetracyclines
Tetracycline is the representative for all tetracyclines and the results
can be applied to doxycycline and minocycline. Certain organisms
may be more susceptible to doxycycline and minocycline than to
tetracycline.
Tetracycline <4 8 >16
 68

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Enterobacteriaceae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Fluoroquinolones
Ciprofloxacin or <1 2 >4
Levofloxacin <2 4 >8
Lomefloxacin or <2 4 >8 Urine only
Norfloxacin <4 8 >16
or <2 4 >8
ofloxacin
Quinolones
Cinoxacin <16 32 >64 Urine only
Folate Pathway Inhibitors
Trimethoprim/ <2/38 - >4/76
sulfamethoxazole
Sulfonamides <256 - >512 Urine only
Sulfisoxazole can
be used to
represent any of
the currently
available
sulfonamide
preparations.
Trimethoprim <8 - >16 Urine only
Phenicols
Chloramphenicol <8 16 >32 Not routinely
tested against
organisms
isolated from the
urinary tract.
 69

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Enterobacteriaceae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Nitrofurantoins
Nitrofurantoin <32 64 >128 Urine only

Notes:
1. For isolates of Salmonella and Shigella sp., only
ampicillin, a fluoroquinolone, and
trimethoprim/sulfamethoxazole should be tested and
reported routinely. In addition, chloramphenicol and a third-
generation cephalosporin should be tested and reported for
extraintestinal isolates of Salmonalla sp.
 70

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Enterococcus sp.
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Beta-lactams – Penicillins
Ampicillin is the class representative for ampicillin and amoxicillin.
Ampicillin results may be used to determine susceptibility to
amoxicillin/clavulanic acid and ampicillin/sulbactam among non-beta-
lactamase producing enterococci.

Penicillin susceptibility may be used to predict the susceptibility to

ampicillin, amoxicillin, ampicillin/sulbactam, amoxicillin/clavulanic
acid, piperacillin, and piperacillin/tazobactam for non-beta-lactamase
producing enterococci.

The “susceptible” category for penicillin or ampicillin implies the

need for high-dose therapy for serious enterococcal infections.
Enterococcal endocarditis requires combined therapy with high-dose
penicillin (or high-dose ampicillin, or vancomycin or teicoplanin) plus
gentamicin or streptomycin for bactericidal action.

Because ampicillin or penicillin resistance among enterococci due to

beta-lactamase production is not reliably detected using routine
dilution methods, a direct, nitrocefin-based beta-lactamase test is
recommended for blood and cerebrospinal fluid isolates. A positive
beta-lactamase test predicts resistance to penicillin, as well as
amino-, carboxy-, and ureidopenicillins.
Penicillin or <8 - >16
Ampicillin <8 - >16
Glycopeptides
Vancomycin <4 8-16 >32 When testing
vancomycin,
plates should be
held a full 24
 71

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Enterococcus sp.
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
hours for
accurate
detection of
resistance. For
isolates with
vancomycin MICs
of 8-16 ug/ml,
perform additional
susceptibility
tests.
Teicoplanin <8 16 >32 Investigational
drug
Aminoglycosides – high-level screening test (see note 2)

Gentamicin <500 >500 Synergy Screen

Streptomycin <1000 >1000 Synergy Screen

Macrolides – not routinely reported on isolates from the urinary tract.

Erythromycin <0.5 1-4 >8
Tetracyclines
Tetracycline is the representative for all tetracyclines and the results
can be applied to doxycycline and minocycline. Certain organisms
may be more susceptible to doxycycline and minocycline than to
tetracycline.
Tetracycline <4 8 >16
Doxycycline <4 8 >16
Minocycline <4 8 >16
 72

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Enterococcus sp.
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Fluoroquinolones
Ciprofloxacin <1 2 >4 Urine only
Levofloxacin <2 4 >8 Urine only
Norfloxacin <4 8 >16 Urine only
Phenicols
Chloramphenicol <8 16 >32 Not routinely
tested and
reported against
organisms
isolated from the
urinary tract.
Ansamycins
Rifampin <1 2 >4 Rifampin should
not be used alone
for
chemotherapy.
Nitrofurantoins
Nitrofurantoin <32 64 >128 Urine only

Notes:

1. For Enterococcus sp., cephalosporins, aminoglycosides

(except for high-level resistance screening),
clindamycin, and trimethoprim/sulfamethoxazole may
appear active in vitro but are not effective clinically, and
isolates should not be reported as susceptible.
 73

2. Synergy between ampicillin, penicillin, or vancomycin

and an aminoglycoside can be predicted for enterococci
by using a high-level aminoglycoside (gentamicin and
streptomycin) screening test. Other aminoglycosides
need not be tested, because their activities against
enterococci are not superior to gentamicin and
streptomycin.

3. Because of limited alternatives, chloramphenicol,

erythromycin, tertracycline (or doxycycline or
minocycline), and rifampin may be tested for
vancomycin-resistant enterococci (VRE), and
consultation with an infectious disease practitioner is
recommended.

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Pseudomonas aeruginosa and Other Non-
Enterobacteriaceae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Beta-lactams - Penicillins
Mezlocillin or <64 - >128 For P. aeruginosa
<16 32-64 >128 For other non-
Ticarcillin <64 - >128 Enterics
<16 32-64 >128 For P. aeruginosa
Piperacillin <64 - >128 For other non-
<16 32-64 >128 Enterics
For P. aeruginosa
For other non-
Enterics
Carbenicillin <128 256 >512 For P. aeruginosa
<16 32 >64 For other non-
Enterics
Urine Only
 74

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Pseudomonas aeruginosa and Other Non-
Enterobacteriaceae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Beta-lactams/Beta lactamase Inhibitor Combinations
Ticarcillin/ <64/2 - >128/2 For P. aeruginosa
clavulanic acid <16/2 32/2- <16/2 For other non-
64/2 Enterics
Ampicillin/ <8/4 16/8 >32/16 May be reported
sulbactam for Acinetobacter
sp. resistant to
other agents
Piperacillin/ <64/4 - >128/4 For P. aeruginosa
tazobactam <16/4 32/2- >128/4 For other non-
64/2 Enterics to other
agents
Beta-lactams - Cephems (including cephalosporins I, II, III, and
IV) – (Parenteral)

Ceftazidime <8 16 >32

Cefepime <8 16 >32
Cefoperazone <16 32 >64
Cefotaxime or <8 16-32 >64
Ceftriaxone <8 16-32 >64
Ceftizoxime <8 16-32 >64 Urines only
Moxalactam <8 16-32 >64 May be indicated
for testing of S.
maltophilia
Carbapenems
Imipenem or <4 8 >16
Meropenem <4 8 >16
 75

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Pseudomonas aeruginosa and Other Non-
Enterobacteriaceae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Monobactams
Aztreonam <8 16 >32
Aminoglycosides
Gentamicin <4 8 >16
Amikacin <16 32 >64
Netilmicin <8 16 >32
Tobramycin <4 8 >16
Tetracyclines
Tetracycline is the representative for all tetracyclines and the
results can be applied to doxycycline and minocycline. Certain
organisms may be more susceptible to doxycycline and minocycline
than to tetracycline.
Tetracycline <4 8 >16 May be indicated
for primary testing
of some
Pseudomonas
sp. (other than P.
aeruginosa),
S. maltophilia &
Acinetobacter sp.
Fluoroquinolones
Ciprofloxacin <1 2 >4
 76

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Pseudomonas aeruginosa and Other Non-
Enterobacteriaceae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Levofloxacin or <2 4 >8 Urine only
Lomefloxacin <2 4 >8
or <2 4 >8
Ofloxacin or <4 8 >16
Norfloxacin
Folate Pathway Inhibitors
Trimethoprim/ <2/38 - >4/76 May be indicated
sulfamethoxazole for primary testing
of some
Pseudomonas
sp. (other than P.
aeruginosa),
S. maltophilia &
Acinetobacter sp.
Sulfonamides <256 - >512 Urine only
Sulfisoxazole can
be used to
represent any of
the currently
available
sulfonamide
preparations.
 77

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Pseudomonas aeruginosa and Other Non-
Enterobacteriaceae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Phenicols
Chloramphenicol <8 16 >32 Not routinely
tested against
organisms
isolated from the
urinary tract.
May be indicated
for primary testing
of some
Pseudomonas
sp. (other than P.
aeruginosa),
S. maltophilia &
Acinetobacter sp.

Notes:

1. Non-Enterobacteriaceae include Acinetobacter sp.,

Stenotrophomonas maltophilia, Pseudomonas sp., and
other
non-fastidious, glucose-nonfermenting, gram-negative
bacilli

2. The susceptibility of Pseudomonas aeruginosa isolated

from patients with cystic fibrosis can be reliably
determined, but may require extended incubation up to
24 hours.
 78

3. P. aeruginosa may develop resistance during prolonged

therapy with all antibiotics. Therefore, isolates that are
initially susceptible may be come resistant within three
to four days after initiation of therapy. Testing of repeat
isolated may be warranted.

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Streptococcus pneumoniae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Beta-lactams – Penicillins
A pneumococcal isolate that is susceptible to penicillin can be
considered susceptible to ampicillin, amoxicillin, amoxicillin/clavulanic
acid, ampicillin/sulbactam, cefaclor, cefdinir, cefepime, cefetamet,
cefixime, cefotaxime, cefprozil, ceftibuten, ceftriaxone, cefuroxime,
cefpodoxime, ceftizoxime, imipenem, and loracarbef. Testing of
ampicillin, ampicillin/sulbactam, ceftibuten, cefetamet, ceftizoxime,
and cefixime against penicillin-intermediate or penicillin-resistant
isolates is not recommended, because reliable interpretive criteria for
those agents with S. pneumoniae are not available. Physicians
should be informed that clinical response rates with these agents
might be lower in strains that are not susceptible to penicillin.
<0.06 If penicillin result
Penicillin 0.12-1 >2 is intermediate or
resistant,
cefotaxime,
ceftriaxone, and
meropenem MICs
should also be
reported.

High doses of
intravenous
 79

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Streptococcus pneumoniae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
penicillins (i.e., 2
million units every
4 hours in adults
with normal renal
function) or
similarly ampicillin
are effective in
treating
pneumococcal
pneumonia due to
strains in the
intermediate
category.
Amoxicilllin <2 4 >8
Beta-lactams – Cephems (Parenteral) (including
cephalosporins I, II, III, and IV)
Cefepime <0.5 1 >2 In standard
dosages,
cefepime is
effective in
treating
pneumococcal
pneumonia
caused by
“intermediate-
category”
strains.
Cefotaxime <0.5 1 >2 See note 4
Ceftriaxone (CSF) <0.5 1 >2 See note 4
Ceftriaxone (non- <1.0 2 >4
 80

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Streptococcus pneumoniae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
CSF)
Cefuroxime <0.5 1 >2
sodium
Beta-lactams – Cephems (Oral) (including cephalosporins I, II,
III, and IV)
Cefaclor <1 2 >4
Cefdinir <0.5 1 >2
Cefpodoxime <0.5 1 >2
Cefproxil <2 4 >8
Cefuroxime axetil <1 2 >4
(oral)
Carbapenems
Meropenem <0.25 0.5 >1
Imipenem <0.12 0.25-0.5 >1
Glycopeptides
Vancomycin <1 No S.
pneumoniae
strain with
vancomycin MIC
>1 ug/ml has
been observed.
Submit such
strains to a
reference lab.

Macrolides – not routinely reported on isolates from the urinary tract.

Erythromycin <0.25 0.5 >1
 81

Azithromycin <0.5 1 >2

Clarithromycin <0.25 0.5 >1
Tetracyclines
Tetracycline <2 4 >8
Fluoroquinolones
Levofloxacin <2 4 >8
Ofloxacin <2 4 >8 Ofloxacin-
susceptible S.
pneumoniae will
also be
susceptible to
levofloxacin.
Lincosamides
Clindamycin <0.25 0.5 >1 Not routinely
tested and
reported against
organisms
isolated from the
urinary tract.
Folate Pathway Inhibitors
Trimethoprim/ <0.5/9.5 1/19-2/38 >4/76
sulfamethoxazole
Phenicols
Chloramphenicol <4 - >8 Not routinely
tested against
organisms
isolated from the
urinary tract.
 82

Ansamycins
Rifampin <1 2 >4 Rifampin should
not be used alone
for
chemotherapy.

Notes:

1. Results of testing with penicillin, cefotaxime, cetriaxone,

meropenem, and vancomycin should be reported
routinely with blood and CSF isolates of S. pneumoniae
recovered from patients with life-threatening infections
(i.e., meningitis, bacteremia).

2. Medium used: cation-adjusted Mueller-Hinton broth

with lysed horse blood (2-5% v/v)

3. Incubation: 35C, ambient air, 20-24 hours

4. In standard dosages, cefotaxime or ceftriaxone are

effective in treating pneumococcal pneumonia caused
by “intermediate-category” strains. However, when
recovered from patients with meningitis, strains in the
intermediate category may require therapy with
maximum doses of cefotaxime or ceftriaxone.
 83

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Streptococcus sp. other than Streptococcus pneumoniae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Beta-lactams – Penicillins
A streptococcal isolate that is susceptible to penicillin can be
considered susceptible to ampicillin, amoxicillin, amoxicillin/clavulanic
acid, ampicillin/sulbactam, cefaclor, cefazolin, cephalothin, cefdinir,
cefepime, cefotaxime, cefprozil, ceftibuten (Group A streptococci
only), ceftriaxone, cefuroxime, cefpodoxime, ceftizoxime, imipenem,
meropenemand loracarbef, and need not be tested against those
agents.
Penicillin or <0.12 - - Strains of beta-
Ampicillin <0.25 - - hemolytic
(beta only) streptococci with
penicillin MICs of
greater than 0.12
ug/ml or ampicillin
MICs of greater
than 0.25 ug/ml
have not been
observed; submit
such strains to a
reference lab.
Amoxicilllin <2 4 >8
Beta-lactams – Cephems (Parenteral) (including
cephalosporins I, II, III, and IV)
Cefepime <0.5 1 >2 In standard
dosages,
cefepime is
effective in
treating
pneumococcal
pneumonia
 84

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Streptococcus sp. other than Streptococcus pneumoniae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
caused by
“intermediate-
category”
strains.
Cefotaxime or <0.5 1 >2 In standard
Ceftriaxone <0.5 1 >2 dosages,
cefotaxime or
ceftriaxone are
effective in
treating
pneumococcal
pneumonia
caused by
“intermediate-
category”
strains. However,
when recovered
from patients with
meningitis, strains
in the
intermediate
category may
require therapy
with maximum
doses of
cefotaxime or
ceftriaxone.
Cefuroxime <0.5 1 >2
sodium
 85

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Streptococcus sp. other than Streptococcus pneumoniae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Beta-lactams – Cephems (Oral) (including cephalosporins I, II,
III, and IV)
Cefaclor <1 2 >4
Cefdinir <0.5 1 >2
Cefpodoxime <0.5 1 >2
Cefproxil <2 4 >8
Cefuroxime axetil <1 2 >4
(oral)
Carbapenems
Meropenem <0.25 0.5 >1
Imipenem <0.12 0.25-0.5 >1
Glycopeptides
Vancomycin <1 No Streptococcus
strain with
vancomycin MIC
>1 ug/ml has
been observed.
Submit such
strains to a
reference lab.
Macrolides – not routinely reported on isolates from the urinary tract.
Erythromycin <0.25 0.5 >1
Azithromycin <0.5 1 >2
Clarithromycin <0.25 0.5 >1
Tetracyclines
Tetracycline <2 4 >8
 86

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Streptococcus sp. other than Streptococcus pneumoniae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Fluoroquinolones
Levofloxacin <2 4 >8
Ofloxacin <2 4 >8 Ofloxacin-
susceptible
Streptococci will
also be
susceptible to
levofloxacin.
Lincosamides
Clindamycin <0.25 0.5 >1 Not routinely
tested and
reported against
organisms
isolated from the
urinary tract.
Folate Pathway Inhibitors
Trimethoprim/ <0.5/9.5 1/19-2/38 >4/76
sulfamethoxazole
Phenicols
Chloramphenicol <4 - >8 Not routinely
tested against
organisms
isolated from the
urinary tract.
 87

Minimum Inhibitory Concentration (MIC) Interpretive Standards

(ug/ml) for Streptococcus sp. other than Streptococcus pneumoniae
Antimicrobial Suscept Interme Resist Comments
Agent ible diate ant
Ansamycins
Rifampin <1 2 >4 Rifampin should
not be used alone
for
chemotherapy.

Notes:

1. Results of testing with penicillin, cefotaxime, cetriaxone,

meropenem, and vancomycin should be reported
routinely with blood and CSF isolates of S. pneumoniae
recovered from patients with life-threatening infections
(i.e., meningitis, bacteremia).

5. Medium used: cation-adjusted Mueller-Hinton broth

with lysed horse blood (2-5% v/v)

6. Incubation: 35C, ambient air, 20-24 hours

88
89
90
91
92
93
 94

MIC Panel Keys

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC Interpretive
Standards Criteria’ interpret the result as susceptible,
intermediate or resistant.

Organism tested: Staphylococcus aureus

Interpretation
Antibiotic Tested MIC (susceptible, intermediate,
Result resistant)
> 0.25
Penicillin Resistant
mcg/ml
4.0
Oxacillin Resistant
mcg/ml
8.0
Erythromycin Resistant
mcgml
8.0
Cefazolin Susceptible
mcg/ml
4.0
Clindamycin Resistant
mcg.ml
< 4.0
Gentamicin Susceptible
mcg/ml
32.0
Vancomycin Resistant
mcg/ml

4. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
No, these results do not follow characteristic antimicrobial
susceptibility patterns (antibiogram information and
examples of predictable susceptibility patterns).
 95

Based on the oxacillin result, this is a MRSA. Confirmation

testing may be required. If this is a MRSA, all beta-lactam
antibiotics are reported as resistant (cefazolin is reported
as resistant)

Vancomycin – based on this susceptibility this is a VRSA

which is very rare. Confirmation testing must be
performed.
 96

MIC Panel Keys

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC Interpretive
Standards Criteria’ interpret the result as susceptible,
intermediate or resistant.

Organism tested: Staphylococcus sp. coagulase negative

Interpretation
Antibiotic Tested MIC (susceptible, intermediate,
Result resistant)
> 0.25
Penicillin Resistant
mcg/ml
4.0
Oxacillin Resistant
mcg/ml
8.0
Erythromycin Resistant
mcgml
8.0
Cefazolin Susceptible
mcg/ml
4.0
Clindamycin Resistant
mcg.ml
< 4.0
Gentamicin Susceptible
mcg/ml
32.0
Vancomycin Resistant
mcg/ml

1. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
No, these results do not follow characteristic antimicrobial
susceptibility patterns (antibiogram information and
examples of predictable susceptibility patterns).
 97

Based on the oxacillin result, all beta-lactam antibiotics are

reported as resistant (cefazolin is reported as resistant)

Vancomycin – based on this susceptibility this is a

vancomycin-resistant Staphylococci which is very rare.
Confirmation testing must be performed.
 98

MIC Panel Keys

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC Interpretive
Standards Criteria’ interpret the result as susceptible,
intermediate or resistant.

Organism tested: Enterococcus sp.

Interpretation
Antibiotic Tested MIC Result (susceptible, intermediate,
resistant)
> 16.0
Ampicillin Resistant
mcg/ml
32.0
Vancomycin Resistant
mcg/ml
Gentamicin
Resistant
Synergy
Streptomycin
Resistant
Synergy

1. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
All results follow characteristic antimicrobial susceptibility
patterns (antibiogram information and examples of
predictable susceptibility patterns) if this is a VRE.
Confirmation of VRE is usually performed.
 99

MIC Panel Keys

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC Interpretive
Standards Criteria’ interpret the result as susceptible,
intermediate or resistant.

Organism tested: Escherichia coli

Interpretation
Antibiotic Tested MIC Result (susceptible, intermediate,
resistant)
> 64.0
Ampicillin Resistant
mcg/ml
Ampicillin/ < 8/4
Susceptible
Sulbactam mcg/ml
Gentamicin 4.0 mcg/ml Susceptible
> 32.0
Cefazolin Resistant
mcg/ml
> 32.0
Cefoxitin Resistant
mcg/ml
< 8.0
Cefotaxime Susceptible
mcg/ml
> 64.0
Amikacin Resistant
mcg/ml
< 4.0
Imipenem Susceptible
mcg/ml

1. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
No, all results do not follow characteristic antimicrobial
susceptibility patterns (antibiogram information and
examples of predictable susceptibility patterns).
 100

Most E. coli isolates are susceptible to amikacin. Also, if

gentamicin is susceptible, then amikacin should also be
susceptible.

Most E. coli isolates are susceptible to cefotaxime. This is

possible, but a resistant strain.
 101

MIC Panel Keys

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC Interpretive
Standards Criteria’ interpret the result as susceptible,
intermediate or resistant.

Organism tested: Klebsiella pneumoniae

Interpretation
Antibiotic Tested MIC Result (susceptible, intermediate,
resistant)
> 64.0
Ampicillin Resistant
mcg/ml
Ampicillin/ 16/8
Intermediate
Sulbactam mcg/ml
> 16.0
Gentamicin Resistant
mcg/ml
Cefazolin 8.0 mcg/ml Susceptible
< 4.0
Cefoxitin Susceptible
mcg/ml
64.0
Cefotaxime Resistant
mcg/ml
< 16.0
Amikacin Susceptible
mcg/ml
> 128.0
Piperacillin Resistant
mcg/ml

1. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
No, all results do not follow characteristic antimicrobial
susceptibility patterns (antibiogram information and
examples of predictable susceptibility patterns).
 102

Most K. pneumoniae isolates are susceptible to

cefotaxime. Also, if cefazolin and cefoxitin is susceptible,
then cefotaxime should also be susceptible.

Most K. pneumoniae isolates are susceptible to

gentamicin. This is possible, but a resistant strain.
 103

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC Interpretive
Standards Criteria’ interpret the result as susceptible,
intermediate or resistant.

Organism tested: Group B Streptococcus .

Interpretation
Antibiotic Tested MIC Result (susceptible, intermediate,
resistant)
< 0.12
Penicillin Susceptible
mcg/ml
Vancomycin 4.0 mcg/ml Not Susceptible
> 4.0
Erythromycin Resistant
mcg/ml

1. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
No, all results do not follow characteristic antimicrobial
susceptibility patterns (antibiogram information and
examples of predictable susceptibility patterns).

No Group B Streptococcus has had a vancomycin MIC

>1.0 mcg/ml.
 104

MIC Panel Keys

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC Interpretive
Standards Criteria’ interpret the result as susceptible,
intermediate or resistant.

Organism tested: Streptococcus pneumoniae

Interpretation
Antibiotic Tested MIC (susceptible, intermediate,
Result resistant)
> 2.0
Penicillin Resistant
mcg/ml
4.0
Oxacillin Resistant
mcg/ml
2.0
Erythromycin Resistant
mcgml
< 0.5
Cefotaxime Susceptible
mcg/ml
< 0.5
Ceftriaxone Susceptible
mcg/ml
< 0.25
Meropenem Susceptible
mcg/ml
2.0
Vancomycin Not Susceptible
mcg/ml

5. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
No, these results do not follow characteristic antimicrobial
susceptibility patterns (antibiogram information and
examples of predictable susceptibility patterns).
 105

No Streptococcus pneumoniae has had a vancomycin MIC

>1.0 mcg/ml.

Based on the antibiogram data, approximately 25% of

Streptococcus pneumoniae isolates are resistant to
erythromycin. This is certainly possible, but is more
resistant than most isolates.
106
107
108
109
 110

Urine MIC Panel Keys

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC Interpretive
Standards Criteria’ interpret the result as susceptible,
intermediate or resistant.

Organism tested: Escherichia coli from Urine

Interpretation
Antibiotic Tested MIC (susceptible, intermediate,
Result resistant)
32.0
Ampicillin Resistant
mcg/ml
Ampicillin/ < 8/4
Susceptible
Sulbactam mcg/ml
4.0
Gentamicin Susceptible
mcg/ml
< 4.0
Cefazolin Susceptible
mcg/ml
8.0
Cefoxitin Susceptible
mcg.ml
Trimethoprim/
< 2/38
Sulfamethoxaz Susceptible
mcg/ml
ole
< 16.0
Nitrofurantoin Susceptible
mcg/ml
< 4.0
Norfloxacin Susceptible
mcg/ml

6. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
 111

All results follow characteristic antimicrobial susceptibility

patterns (antibiogram information and examples of
predictable susceptibility patterns)
 112

Urine MIC Panel Keys

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC Interpretive
Standards Criteria’ interpret the result as susceptible,
intermediate or resistant.

Organism tested: Klebsiella pneumoniae from Urine

Interpretation
Antibiotic Tested MIC (susceptible, intermediate,
Result resistant)
> 64.0
Ampicillin Resistant
mcg/ml
Ampicillin/ < 8/4
Susceptible
Sulbactam mcg/ml
4.0
Gentamicin Susceptible
mcg/ml
16
Cefazolin Intermediate
mcg/ml
8.0
Cefoxitin Susceptible
mcg/ml
Trimethoprim/
< 2/38
Sulfamethoxaz Susceptible
mcg/ml
ole
< 16.0
Nitrofurantoin Susceptible
mcg/ml
< 4.0
Norfloxacin Susceptible
mcg/ml

7. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
 113

All results follow characteristic antimicrobial susceptibility

patterns (antibiogram information and examples of
predictable susceptibility patterns)
 114

Urine MIC Panel Keys

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC Interpretive
Standards Criteria’ interpret the result as susceptible,
intermediate or resistant.

Organism tested: Proteus mirabilis from Urine

Interpretation
Antibiotic Tested MIC Result (susceptible, intermediate,
resistant)
Ampicillin 8.0 mcg/ml Susceptible
Ampicillin/ < 8/4
Susceptible
Sulbactam mcg/ml
Gentamicin 4.0 mcg/ml Susceptible
< 4.0
Cefazolin Susceptible
mcg/ml
Cefoxitin 8.0 mcg/ml Susceptible
Trimethoprim/
< 2/38
Sulfamethoxaz Susceptible
mcg/ml
ole
> 128.0
Nitrofurantoin Resistant
mcg/ml
>16
Tetracycline Resistant
mcg/ml
< 4.0
Norfloxacin Susceptible
mcg/ml

8. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
 115

All results follow characteristic antimicrobial susceptibility

patterns (antibiogram information and examples of
predictable susceptibility patterns)
 116

Urine MIC Panel Keys

Interpretation of MICs – use the provided MIC panels:

Record the MIC result and using the provided ‘MIC Interpretive
Standards Criteria’ interpret the result as susceptible,
intermediate or resistant.

Organism tested: Pseudomonas aeruginosa from Urine

Interpretation
Antibiotic Tested MIC Result (susceptible, intermediate,
resistant)
>16.0
Gentamicin Resistant
mcg/ml
< 8.0
Amikacin Susceptible
mcg/ml
Ciprofloxacin 1.0 mcg/ml Susceptible
< 4.0
Norfloxacin Susceptible
mcg/ml
Ceftazidime 8.0 mcg/ml Susceptible
Aztreonam 16 mcg/ml Intermediate
> 128.0
Mezlocillin Resistant
mcg/ml
< 4.0
Imipenem Susceptible
mcg/ml

9. Using the provided ‘Clinical Microbiology Antibiogram’ and

known predictable patterns of susceptibility information, do
these results follow characteristic antimicrobial susceptibility
patterns? Explain.
All results follow characteristic antimicrobial susceptibility
patterns (antibiogram information and examples of
predictable susceptibility patterns)