Laboratory Methods for Antimicrobial i.

Principles
Susceptibility Testing  Evaluating antimicrobial susceptibility
1. Collection and Transport 1. Measuring the activity of one or more antimicrobial
2. Direct Detection agents against a bacterial isolate.
3. Cultivation and Identification 2. Detect the presence of a specific resistance
4. Antimicrobial Susceptibility & Resistance mechanism in a bacterial isolate.
3. Measure antimicrobial-organism interactions.
5 antibiotics (least) against a bacteria in
I. Goal and Limitations antimicrobial susceptibility testing
A. Goal intermediate – prescribed
 Determine whether the organism is capable of
expressing resistance and the extent of its acquired ii. Measuring Antimicrobial Activity
resistance. A. Conventional Susceptibility Testing
1. Broth dilution
B. Standardization and Its Limitations 2. Agar dilution
i. Standardization 3. Disc diffusion
 Purpose
o To optimize bacterial growth conditions  Inoculum Preparation
o To maintain antimicrobial activity i. Select 4-5 pure colonies and inoculate them to a broth
o To maintain reproducibility medium or 0.85% saline (NSS)
 The Standardized Components ii. Standardized inoculum using McFarland turbidity
1. Bacterial inoculum size standards (comparable to the density of a bacterial
standard – McFarlan suspension of 1.5 x108 CFU/ml)
2. Growth medium (pH, cation concentration, o Broth dilution → 5 x 105 CFU/mL
supplements and thymidine content) o Agar dilution →1 x 104 CFU/mL
MHA o Disk diffusion → 1.5 x 108 CFU/mL
3. Incubation atmosphere  Nephelometer / Spectrophotometer more precise than
4. Incubation temperature visual adjustment (but visual observation can be used)
usually aerobic
 0.5 McFarland: Mix 0.05 mL of
5. Incubation duration
usually 24 hours 1.175% barium chloride
6. Antimicrobial concentration tested dehydrate (BaCl2•2H2O), with
9.95 mL of 1% sulfuric acid
ii. Limitations (H2SO4).
 Lack of correlation between in vitro test conditions and standard – around 10mL
in vivo setting in antimicrobial susceptibility testing.
o Antibiotic diffusion in tissues and host cell  Selection of Antimicrobial Agents
o Serum protein binding of antimicrobial agents i. Organism identification or group
o Drug interactions and interference o Choose an antimicrobial panel
o Status of patient defense and immune systems o Antimicrobials to which organism is naturally
o Multiple simultaneous illness resistant are excluded.
o Virulence and pathogenicity of infecting ii. Acquired resistance pattern
bacterium o More potent antimicrobials are included
o Site and severity of infection iii. Antimicrobial Susceptibility Testing Method Used
iv. Site of infection - ex. UTI (Nitrofurantoin, Ofloxacin)
v. Availability of Antimicrobial Agent in Formulary
II. Testing Methods vi. Patient group - ex. contraindicated in pediatrics:
fluoroquinolones (impair cartilage dev’t); tetracycline
Factors to Consider When Determining Whether
Testing is Warranted (damages developing teeth)
1. The body site from which the organism is isolated.
e.g. E.coli in stool or blood; S.viridans in throat; Antimicrobial panel bacteria
o Enterobacteriaceae
S.epid isolated in multiple blood cultures
o Pseudomonas aeruginosa and Acinetobacter
2. The presence of other bacteria and the quality of the
spp.
specimen from which the organism was grown. e.g.
o Staphylococcus
mixed culture in urine; K.pneumoniae(if pure)in
o Enterococcus spp.
sputum culture
o Streptococcus spp.
3. The host’s status - e.g. Immunocompromised,
o Haemophilus influenzae
elderly, newborn, patients w/ allergy to certain
o Neisseria gonorrhoeae
antibiotics (penicillin)/erythromycin alternative
1. Broth Dilution 3. Translate MIC into interpretive categories (CLSI)
 Twofold serial dilution series o Breakpoints: susceptible, intermediate or
 Medium and Antimicrobial agents resistance
1. Macrodilution (test tubes → 1-2 ml ) Breakpoint(cut-off)- conc. of antimirobial agent that
2. Microdilution (microtitre trays → 0.05-0.1 ml) coincides with a susceptible or intermadiate MIC
fastidious bacteria – horse blood or sheep’s blood breakpoint for a particular drug
(2-5%)
 Inoculation and Incubation 2. Agar Dilution
Organism Test Inoculu Incubatio Duratio  Medium and Antimicrobial agents
Mediu m Size n n o Doubling dilutions of antimicrobial agent is
m incorporated into single agar plate (6 dilutions /
Enterobacteriace M-H 5x105 35ºC 16-20
a plates)
broth range – 35 hrs
P. aeruginosa to 37  Inoculation and Incubation
N. meningitidis Organism Test Inoculu Incubatio Duratio
M-H 5x105 35ºC 24 hrs Mediu m Size n n
plus 5-7% m
2-5% CO2 Enterobacteriace M-H 1x104 35ºC 16-20
Horse a
hrs
P. aeruginosa
blood
Staphylococcus M-H 1x104 35ºC 24 hrs
plus CO2 5-7%
2%
NaCl
For fastidious organisms MHA can be supplemented w/
5% sheep’s blood

 Medium and Antimicrobial agents

o Doubling dilutions of antimicrobial agent is
incorporated into single agar plate

Test bacteria are “spot”

inoculated into each plate

ipapatak at ikakalat ang

Macrodilution Susceptibility broth
Impractical when several antibiotics must be tested in
one or several isolates
using test tube 1-2 ml
 Reading and Interpretation of Results
side effects for higher volume of antibiotics
zone of inhibition 1. Examine the Controls
o Growth Control
o Sterility Control
2. Record the Minimal Inhibitory concentration
o Plates containing the lowest drug
concentration that inhibited visible bacterial
growth.
3. Translate MIC into interpretive categories
o Breakpoints: Susceptible, Intermediate or
Resistance
RITM and CLSI controls the breakpoints (has zone
of inhibition cheat sheet)

Microdilution Susceptibility
Growth maybe seen as turbidity, a haze or a pellet in the
bottom of the well
using microtiter plates with 0.05 to 0.1ml

 Reading and Interpretation of Results

1. Examine the Controls
o Growth Control (MH Broth and Inoculum)
o Sterility Control (MH Broth)
2. Record the Minimal Inhibitory concentration (MIC)
o Microdilution well containing the lowest drug
concentration that inhibited visible bacterial
growth.
3. Disc Diffusion (Kirby-Bauer) Variable Standard
most commonly used Incubation
cost-effective method Atmosphere Humidified ambient air
 Inoculation and Incubation Temperature 35°C
o Antibiotic disks is place in MH after inoculated Length Disk diffusion: 16-18 hr
with a standardized suspension of bacteria Broth Microdilution: 16-20 hr
Organism Test Inoculum Incubation Duration
Medium Size MHA reading after 34 hrs – false susceptible
E. coli 1.5x108 35ºC; air 16-18 MHA reading too early – false resistant
hrs
B. Commercial Susceptibility Testing
1. Agar Dilution Derivatives
 Spiral Gradient Instrument
o An instrument deposits the
highest concentration of
antibiotic from the center of the
plate to the peripheries
o Bacterial inocula are applied to
the gradient. (inner to outer v
 Reading and Interpretation of Results
concentration)
1. Measure Zone of inhibition for each antibiotic disk
(using caliper)
2. Translate Zone sizes into interpretive categories  E-test (antibiotic gradient in plastic strip)
 Susceptible, Intermediate or Resistant

The MIC is read where growth and inhibition edge

intersects.
 The E-test utilizes a rectangular strip that has been
impregnated with the drug to be studied.
 A lawn of bacteria is spread and grown on an agar
plate, and the E-test strip is laid on top; the drug
diffuses out into the agar, producing an exponential
gradient of the drug to be tested.
 There is an exponential scale printed on the strip.
Mueller-Hinton Agar must be 4mm thick and plates After 24 hours of incubation, an elliptical zone of
should not stacked >5. inhibition is produced and the point at which the
ellipse meets the strip gives a reading for the
Standardization minimum inhibitory concentration (MIC) of the drug.
Variable Standard
Media 2. Automated Systems
Formulation Mueller-Hinton  Vitek 2 (test card and analyzer)
Ca2+, Mg2+ 25 mg/L Ca2+, 12.5 mg/L Mg2+
content
Thymidine content Minimal or absent

pH 7.2-7.4
Organism suspension is introduced into the card by
Agar depth 3-5mm (4 mm; optimal)
automated filling process
Less than 3mm - false sensitive; more than 5mm - false
resistant
diffusion of antibiotic will be wide

Variable Standard
Inoculum Disk diffusion: 1.5 x 108 CFU/mL
Broth microdilution: 5 x 105 CFU/mL
Agar dilution: 1x104 CFU/mL
8:59
C. Antimicrobial Resistance Detection D. Antimicrobial-Organism Interaction
1. Enhancing resistance detection 1. Bactericidal Tests
Test Purpose  Minimal Bactericidal Concentration (MBC)
Oxacillin Agar Detection of staphylococcal o Lowest conc. of antimicrobial agent that kills
Screen resistance to 99.9% of the test bacteria
penicillinase-resistant penicillins o An aliquot from each tube showing inhibition of
Predictor Antimicrobial Agent visible bacterial growth is subcultured to an
Vancomycin Agar Detection of enterococcal enriched agar medium
Screen resistance to o The antimicrobial conc. that resulted in 99.9%
vancomycin reduction is CFU/ml compared with the
Aminoglycoside Detection of acquired organism conc. in the original inoculum.
Screens enterococcal resistance
(Gentamicin) to aminoglycoccides using
gentamicin
Predictor Antimicrobial Agent
Oxacillin Disk Detection of Streptococcus
Screen pneumoniae
resistance to penicillin
“D” Test Differentiate clindamycin
(Macrolide resistance among  Time-Kill Studies
resistance) S. aureus from efflux (msrA or o Method of measuring rate of killing of bacterial
MLSB) isolate by antimicrobial agent by examining
number of viable bacteria remaining at various
 Oxacillin Resistance Test for Staphylococcus intervals after exposure to the agent
 Oxacillin screen plate- MHA w/ 4% NaCl % oxacillin. o A bacterial isolate is exposed to a conc. of
Growth (more than 1 colony is +). Not reliable on oxa- antibiotic in a broth medium. A 1000-fold
R CoagNegStaph. -> testing a surrogate marker of decrease in 24-hour is the bactericidal activity
resistance (i.ecefoxitin) may provide a more accurate
indication of oxacillin resistance
 Cefoxitin serves to induce greater expression of
PBP2a in mecA-containing strains of Staphylococci
 Cefoxitin disk diffusion is now recommended by the
CLSI in oxacillin resistance detection for
Staphylococci
oxacillin - control  Serum Bactericidal Test
o Trough specimen – just before the patient is to
“D” Test receive the next antimicrobial dose
o Peak specimen – the serum antimicrobial
Blunting of clindamycin concentration is highest
zone to give “D” pattern, specimen (serum) of the patient taking the antibiotic
indicates inducible
clindamycin resistance
due to MLSB mechanism

2. Specific resistance
mechanisms
detection
 Phenotypic Method
o Beta-Lactamase Detection by cefinase disk
 N. gonorrhoeae, H. influenzae and o Serumstatic titre is the highest dilution inhibiting
Staphylococcal resistance to penicillin. visibly detectable growth
o Serumcidal titre is the serum dilution that results
Positive in 99.9% reduction in CFU/ml in SBA
(Deep pink)

Negative
(No color change)
Chromogenic
cephalosphorin Test
2. Test for Activity of Antimicrobial Combinations
 Synergy testing
o Synergy
 the activity of the antimicrobial
combination is greater than the activity of
the single drug
o Indifferent
 the activity of combination is no better or
worse that the single drug alone
o Antagonism
 the activity of the combination is less than
activity of single drug alone

Drop Plating
 Label the bottom of the agar plates by dividing them
into fourths with a ruler and marking pen.
 Each serial dilution of the sample will occupy one
quadrant of each plate. Prepare plates in duplicate.
 Pick up 10ul sample with a micropipette 10 µl of
sample is dropped onto the quadrant of one of the
petri plates that have been labeled for that particular
species.
 Repeat this procedure with the duplicate plate.
 Let the drops soak into the media before turning
plates over for incubation.
 Incubate overnight (18-23 hours) at 35-37˚C.
 Remove plates when colonies have developed and
count the dilution which contains 3-30 colonies per 10
µl drop (1x104).
 Viable cell counts are expressed as colony forming
units (CFU)/surface area.

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