ANTIMICROBIAL

TESTING
Terminologies
• Antibiotics
 Substances obtained and purified from other microbial organisms
• Antimicrobial agents
 Collective term
• Bacteriostatic
 Inhibit bacterial growth but do not kill the organisms
• Bactericidal
 Agents that usually kill target organisms
 Antimicrobial Susceptibility Testing
Methods.
• Procedures used to produce antimicrobial susceptibility profiles
and detect resistance to therapeutic agents
• May be categorize based on endpoint use
• Inhibition of growth VS kiling
 Standardization
• It
optimizes bacterial growth conditions so that inhibition of growth can
be attributed to the antimicrobial agent against which the organism is
being tested and is not the result of limitations of nutrient,
temperature, or other environmental conditions that may hinder the
organism’s growth.
• It
optimizes conditions for maintaining antimicrobial integrity and
activity; thus, failure to inhibit bacterial growth can be attributed to
organism associated resistance mechanisms rather than to
environmental drug inactivation.
• It
maintains reproducibility and consistency in the resistance profile of
an organism, regardless of the microbiology laboratory performing the
test.
Standardization
The standardized components of antimicrobial susceptibility testing include:
• Bacterial inoculum size
• Growth medium (most frequently a Mueller-Hinton base)
 pH
 Cation concentration
 Blood and serum supplements
 Thymidine content
• Incubation atmosphere
• Incubation temperature
• Incubation duration
• Antimicrobial concentrations
LIMITATIONS OF STANDARDIZATION
• Antibiotic diffusion into tissues and host cells
• Serum protein binding of antimicrobial agents
• Drug interactions and interference
• Status of patient defense and immune systems
• Multiple simultaneous illnesses
• Virulence and pathogenicity of infecting bacterium
• Site and severity of infection
 Testing Methods (Principle)
• Methods that directly measure the activity of one or more
antimicrobial agents against a bacterial isolate
• Methods that directly detect the presence of a specific resistance
mechanism in a bacterial isolate
• Special methods that measure complex antimicrobial-organism
interactions
 Direct Measure of Antimicrobial Activity
• Conventional susceptibility testing methods such as broth
dilution, agar dilution, and disk diffusion
• Commercial susceptibility testing systems
• Special screens and indicator tests
 When NOT to perform AST:
When the causative organism belongs to a species with predictable
susceptibility to specific drugs.
 Streptococcus pyogenes and Neisseria meningitidis, which are still
generally susceptible to benzylpenicillin.
If the causative organism requires enriched media.
 Haemophilus influenza and Neisseria gonorrhoeae.
In uncomplicated intestinal infections caused by salmonellae (other
than S. typhi or S. paratyphi).
 Antimicrobial treatment of such infections is not justified, even with
drugs showing in vitro activity.
DISK DIFFUSION
DISK DIFFUSION
• Paper discs, impregnated with a specified amount of an
antimicrobial, are placed on agar medium uniformly seeded with
the test organism.
• A concentration gradient of the antimicrobial forms by diffusion
from the disc and the growth of the test organism is inhibited at a
distance from the disc that is related, among other factors, to the
susceptibility of the organism
 General Considerations
• Inoculum preparation
 Use of pure culture
 3 to 5 colonies of the same morphology
 Inoculate into a broth medium allowing the culture to achieve
active growth (3-5 hours of incubation)
 4-5 colonies (16-24 hours old) from an agar plate and suspended
in broth or 0.9% saline solution to achieve a turbid suspension

NOTE: Using an inoculating loop or a cotton swab pick only well-isolated

colonies from the plate to avoid testing mixed cultures. If you do not have well-
isolated colonies, subculture the organism to a fresh plate.
 Inoculum preparation
Two methods for inoculum preparation:
• direct colony suspension
 colonies must not be older than 18–24 hours
 Suspend the colonies in saline or broth
1. All staphylococci
2. Fastidious bacteria that grow unpredictably in broth: e.g., streptococci
• log phase growth.
 used for most organisms that grow rapidly except staphylococci.
 Log phase growth occurs after 2–8 hours incubation.
 General Considerations
Inoculum preparation
 Standard inoculum size
 Comparison of the turbidity of the of the organism suspension with a
turbidity standard.
 MCFARLAND TURBIDITY STANDARD
 1% sulfuric acid and 1.175% barium chloride
 0.5 McFarland standard has a comparable density of a bacterial
suspension of 1.5 x 10^8 CFU

The adjusted suspensions should be used as inoculum within 15 minutes.

• A. What would you do if the suspension of organisms is
too turbid?
• B. What would you do if the suspension is too light for
direct colony suspension?
• C. Whatwould you do if the suspension is too light for
log phase method?
 General Considerations
Testing media
Mueller-Hinton agar or broth
• demonstrates good batch-to-batch reproducibility
• low in sulfonamide, trimethoprim, and tetracycline inhibitors
• supports the growth of most nonfastidious bacteria
 General Considerations
Testing media
• ion concentrations
ORGANISM ION ANTIBIOTIC EFFECT
CONCENTRATION

Pseudomonas  Ca,  Mg aminoglycosides False RESISTANT

aeruginosa
 Ca,  Mg aminoglycosides False SUSCEPTIBLE

 Ca daptomycin False SUSCEPTIBLE

zinc ion carbapenem

 General Considerations
Testing media
• pH of the media
 between 7.2 and 7.4 for accurate results.
 more acidic pH - as aminoglycosides, quinolones, and
macrolides to lose potency
 Some drugs such as penicillin to have increased activity.
 Higher pH can have the opposite effect.
 General Considerations
Testing media
• some fastidious species, supplements must be added
• streptococci do not grow well in or on unsupplemented Mueller-
Hinton media;
 disk diffusion  5% defibrinated sheep blood
 broth dilution  2.5- 5% lysed horse blood
• Haemophilus influenzae Haemophilus test medium (broth
dilution.)
 General Considerations
Testing media
• Neisseria gonorrhoeae  GC agar base medium + growth supplement
• Anaerobes  Brucella agar + laked sheep blood, hemin, and vitamin K.
 agar dilution method(only CLSI recommended method for testing these
species)
• B.fragilis group  Brucella broth + hemin, vitamin K1, and 5% lysed
horse blood
 microbroth dilution method
 General Considerations
Testing media
• Agar depth
 measure 4 mm
 A 9-cm plate requires approximately 25 ml of medium.
*diffusion gradient of the antibiotic will be affected.
 False resistance - too deep
 False susceptibility - agar is not deep enough.
Mueller-Hinton is supplemented with blood,
 Zones of inhibition may be smaller by 2 to 3mm than those obtained
with unsupplemented agar.
 General Considerations
Antimicrobial discs
• 9–10 cm plates  6 or 7 antimicrobial discs
• 150 mm plates  maximum of 12 antimicrobial discs
• 100 mm plates  4-5 antimicrobial discs
• approximately 15 mm from the edge of the plate,
•1 disc placed in the center of the plate
• Disks should not be placed closer than 24 mm (center to center)
 General Considerations
Incubation Conditions
• 35° C±2° C (ambient air) 16–20 hours
• Exceptions:

• Disk diffusion
 Streptococcus spp. and Haemophilus  + 5% carbon dioxide
 Haemophilus spp. are  16–18 hours
 Streptococcus spp.  20–24 hours.
 Staphylococcus spp. and Enterococcus spp. 16–18 hours,
 Staphylococcus spp (oxacillin and vancomycin) full 24 hours
 Enterococcus spp. (vancomycin) full 24 hours
Points to remember about antimicrobial
disks:
• Do not use disks beyond their expiration
date.
• Do not store disks in a frost-free freezer.
• Use FDA cleared products.
• Use disks with the content specified in
NCCLS standards.
• Do not relocate a disk once it has touched
the agar surface.
• Invert and incubate plates with agar side up
Measuring Zones
Measuring Zones

Use transmitted light, rather than reflected

light, when measuring zones for:
• Staphylococci with oxacillin
• Enterococci with vancomycin
BROTH DILUTION
 BROTH DILUTION
• involveschallenging the organism of interest with antimicrobial agents in a
liquid environment.
• each antimicrobial agent is tested using a range of concentration
•a series of doubling dilutions (e.g., 16, 8, 4, 2, 1, 0.5, 0.25 μg/mL)
• For microdilution : the total broth volume is 0.05 to 0.1 mL
• For macrodilution : the broth volumes are usually 1 mL or greater.
MIC
• lowest antimicrobial concentration that completely inhibits
visible bacterial growth, as detected visually or with an
automated or semiautomated method
MIC
• dependent not only on the interaction between the antimicrobial
agent and the organism
 pH and ion concentrations of testing media,
 temperature at which the test system is incubated
 the incubation atmosphere
 the amount of organism used in testing
 the length of time the system incubates.
Minimum inhibitory concentration test
 Dr.T.V.Rao MD
49
MINIMUM BACTERICIDAL
CONCENTRATION TEST

Figure 10.12
 RESULTS OF DISK DIFFUSION
• Susceptible – infection caused by it is likely to respond to treatment
with this antimicrobial, at the recommended dosage.
• Intermediate
 “moderately susceptible” - can be used for treatment at a higher
dosage (e.g. b-lactams)
 “intermediate susceptibility” - more toxic antimicrobial (e.g.
aminoglycoside) that cannot be used at a higher dosage.
• Resistant – organism is expected not to respond to a given
antimicrobial, irrespective of the dosage and of the location of the
infection.
MIC vs. Disk Diffusion Tests
• Streptococcus pneumoniae
 Penicillin—penicillin MIC test when zones of inhibition <20 mm around
oxacillin disks (screening test for penicillin resistance).
 Cefotaxime or ceftriaxone—breakpoints for disk diffusion testing have not
been established
• Viridansstreptococci
 Penicillin—isolates are from normally sterile body sites.
• Staphylococcus species
 Vancomycin—to detect decreased susceptibility to vancomycin since this
cannot be determined using the disk diffusion test.
 Inhibitory Methods for Susceptibility
Testing
Dilution Testing
 Broth Macrodilution
 performed using standard test tubes, and the tubes are
viewed by the unaided eye to determine whether growth is
present.
 No longer used
 Inhibitory Methods for Susceptibility
Testing
Dilution Testing
 Agar dilution method
 antibiotic being tested is incorporated into solid agar in Petri dishes
 isolate being tested is placed on the agar as a single spot, the agar dilution
method can allow the testing of multiple isolates on a single plate.
 With the exception of the Bacteroides fragilis group, agar dilution is the
only standardized method recommended by the CLSI for susceptibility
testing of anaerobe species
 Inhibitory Methods for Susceptibility
Testing
Epsilometer
 plastic strip with a gradient of antibiotic on one side of the strip and a
continuous MIC scale on the opposite side.
 The strip is placed on the surface of an agar plate that has been
inoculated with the isolate. Antibiotic will diffuse into the surrounding
agar in a gradient that corresponds to the gradient on the strip.
 If the isolate is susceptible, an ellipse of inhibition will occur in growth
of the bacterium on the surface of the plate, and the point on the MIC
scale of the strip where the ellipse of inhibition intersects is the MIC
 Inhibitory Methods for Susceptibility
Testing
Epsilometer
 allows the testing of single antibiotics against isolates, which has
particular utility if testing of an isolate against a drug not on the
laboratory’s preconstructed antibiotic panel is necessary
 if testing of one or a few drugs against fastidious species that require
special media is required (e.g., Haemophilus influenzae).
Reading E-tests Ciprofloxacin for
Yersinia pestis

Resistant > 4 ug/ml

Intermediate 1-4 ug/ml

Susceptible < 1

Dr.T.V.Rao MD
Upper reading

61
 Common interpretation problems
Problems with E-test reading

Dr.T.V.Rao MD
62
 Direct Test of β-Lactamase
• detectingpenicillinase enzymes in gram-positive and a few gram-
negative species
• Three types
 Acidometric
 Iodometric
 Chromogenic – most sensitive
 Direct Test of β-Lactamase
• Reagent – cephalosporin (nitrocefin)
• Principle:
organism is producing β-lactamase, hydrolysis of the
chromogenic cephalosporin will occur and the disk color will change
from yellow to red, usually within a minute; (up to 15 mins)
• indicates
that the organism will not be inhibited by a penicillinase-
susceptible penicillin.
 D-ZONE APPROXIMATION TEST
• forpregnant women who are colonized with group B
streptococci and who are allergic to penicillin
• resistant
to erythromycin but susceptible to clindamycin
should be tested for “inducible” resistance to clindamycin
mediated by the erm gene
Reference
• Henry’s Clinical Dx and Mgt. by Lab Mtds.
 Chapter 58 In Vitro Testing of antimicrobial Agents

• Bailey’s and Scotts

 Chapter 12 Lab Mtds and Strategies for Antimicrobial Susceptibility Testing

• Manual of Antimicrobial Susceptibility Testing

• WHO Basic Bacteriologic Procedures