Exercise Number 5

Antimicrobial Susceptibility Test Diffusion Test Procedures

MATTHEW EARL G. MALUMAY | BATCH HILUSA 2022 | IDCM

OUTLINE Antimicrobial Susceptibility Testing

I. Antimicrobial Susceptibility Testing Antimicrobial Susceptibility Testing (AST)
II. Disc Diffusion Method  Significant bacteria isolated from clinical
III. Resistance to Antimicrobial Drugs samples; guide the clinician on which
IV. Origin of Drug Resistance antimicrobial for patient management and
V. Cross Resistance generated susceptibility pattern data for
VI. Limitation of Drug Resistance suspected pathogen.
VII. CLINICAL IMPLICATIONS OF DRUG
 Check the effectiveness of drug against
RESISTANCE: Staphylococci
microorganisms and to select the best drug
VIII. CLINICAL IMPLICATIONS OF DRUG
RESISTANCE: Gram-Negative Enteric that acts against the microorganisms.
Bacteria Right Microbe →Right Drug →Right Cure
IX. Antimicrobial Activity in Vitro
X. Factors Affecting Antimicrobial Activity Methods of Antimicrobial Susceptibility Testing:
 Disc Diffusion Method
REFERENCE o Agar or solid media cultured with
lawn of isolate and impregnated with
Laboratory Manual in Microbiology and
antibiotic discs.
Parasitology Iloilo Doctors College of Medicine
o Plate is incubated for the organism to
Jawetz, Melnick and Adelberg’s Medical grow and antimicrobial to diffuse into
Microbiology 27th Edition Karen Carrol Steohen the agar.
Morse Timothy Mietzner Steve Miller  Serial Dilution
o Organism is tested against a serial
increase in concentration of
antimicrobial in solid or broth media.
o Turbidity –sign of microbial growth on
broth medium and concentration of
antimicrobial without turbidity is
noted.
 Combination of Increasing Amount of
Antibiotic impregnated in a Strip
o Least amount of antibiotic that exhibits
inhibition of growth is taken.

Disc Diffusion Method

Disc Diffusion Method by Kirby Bauer
 principled after regression line graph, the
higher the antimicrobial concentration, the
smaller the zone of inhibition.
 To generate a dynamically changing gradient
of antibiotic concentrations.

Inhibition Zone Edge –formed at a critical time

where the concentration of the antibiotic to inhibit
the organism before it reaches cell (critical) mass.

Factors Standardized on Disc Diffusion Method:

 Depth of Agar
o Thick Agar →Slow Diffusion →Tested
Organism grows Faster
≈FALSE RESISTANT

Matthew Earl G. Malumay MD-SOON IDCM 1

 Antimicrobial Susceptibility Test Diffusion Test Procedures
o 4 mm –standard depth of agar.
 Concentration of Antibiotic
o Indicated in CLSI Manual and followed for
regression graph.
 Inoculum Size
o 0.5 McFarland Nephelometer –standard
inoculum size →1.5 x 108 bacterial
suspension/mL.
 Culture Media
o Mueller Hinton Agar [Recommended]
o Sheep Blood Agar or Horse Blood
[Fastidious Organisms]
 Control Strains
o American Type Culture Collection (ATCC)
–quality control testing of reagents,
materials and supplied to conduct
microbial procedures.
o Escherichia coli ATCC 25922
o Pseudomonas aeruginosa ATCC 27853
o Staphylococcus aureus ATCC 29213
o Haemophilus influenza ATCC 49766
PURPOSE:
To determine the sensitivity of commonly
isolated, rapidly growing pathogens,
Staphylococcus aureus and Enterobateriaceae
Family to antimicrobial agents.
To guide the clinician in selecting the best
antibiotic agent for the patient.
To control the use of inappropriate antibiotic in
clinical practice.
MATERIALS:
Inoculating Needle/Loop
Saline Solution
Antibiotic Disc
Turbidity Standard
Cotton Swab Forceps
Mueller Hinton Agar (MHA) Plates
PROCEDURE:
1. Fish isolated colonies using inoculating loop of
morphological type suspended directly into
saline. Repeat this procedure until the
turbidity of suspension matches on standard
prepared by adding 0.5 mL if 0.048 Barium
Chloride to 99.5 mL of 0.36 N Sulfuric Acid.
2. Dip a sterile swab on applicator stick into
standardized suspension. Remove excess fluid
by pressing and rotating the swab against the
side of the tube above the fluid level. Streak
the swab evenly in three directions over the
Matthew Earl G. Malumay MD-SOON IDCM
entire surface of the agar plate to obtain
uniform inoculum. Allow the plate to dry for
3-5 minutes (not more than 15 minutes).
3. Within 15 minutes, after the plates are
inoculated, apply antibiotic impregnated discs
to the surface of the inoculated plates with
sterile forceps. Gently press down all discs
onto the agar with forceps or inoculating loop
to ensure complete contact with the agar
surface. Special arrangements of the discs
should not be closer than 15 mm to edge of
the plate and far enough to prevent
overlapping zone of inhibition (at least 20 mm
apart).
4. Within 15 minutes after the discs are applied,
the plates are inverted and placed in the
incubator at 35 degrees Celsius.
5. Within 16-18 hours of incubation, examine
the plates and measure to the nearest whole
millimeter the zone of complete inhibition.
Resistance to Antimicrobial Drugs
Sensitivity –zone of inhibition surrounding the
antibiotic disc.
Resistance –no zone of inhibition surrounding the
antibiotic disc.
Mechanisms of Microorganisms that exhibit Drug
Resistance:
 Microorganisms produce Enzymes that
destroy the active drug.
o Staphylococci resistant to penicillin G
produce B lactamase that destroys the
drug.
o Gram (-) bacteria resistant to
aminoglycosides (by plasmid) produce
adenylating, phosphorylating or
acetylating enzymes that destroy the
drug.
 Microorganisms change Permeability to the
drug.
o Tetracyclines accumulate in susceptible
bacteria but not in resistance bacteria.
o Streptococci have natural permeability
barrier to aminoglycosides.
o Resistance to Amikacin and
Aminoglycosides has lack of permeability
to the drugs caused by an outer
membrane change that impairs active
transport into the cell.
 Microorganisms develop an Altered Structural
Target for the Drug.
o Erythromycin-resistant organisms have
altered receptor on 50S subunit of
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 Antimicrobial Susceptibility Test Diffusion Test Procedures
ribosome →Methylation of 23S ribosomal
RNA.
o Resistance to penicillin and cephalosporins
has lost or altered PBPs.
 Microorganisms develop an Altered Metabolic
Pathway that bypasses the reaction inhibited
by the drug.
o Sulfonamide-resistant bacteria do not
require PABA but mammalian cells require
preformed folic acid.
 Microorganisms develop an Altered Enzyme
that can still perform its metabolic function
but is much less affected by the drug.
o Dihydrofolic acid reductase is inhibited in
trimethoprim-resistant bacteria less
efficiently than in trimethoprim-susceptible
bacteria.
 Microorganisms can develop Efflux Pumps
that transport the antibiotics out of the cell.
o Gram (+) and (-) developed efflux pumps
for tetracyclines, macrolides,
fluoroquinolone and B-lactam agents.
Resistance to Antimicrobial Drugs
 Nongenetic Origin of Drug Resistance
o Active replication of bacteria is required
for antibacterial drug actions.
o Consequently, microorganisms that are
metabolically inactive (nonmultiplying) are
phenotypically resistant to drugs.
o Microorganisms lose specific target
structure for a drug for several
generations and be resistant.
o Microorganisms infect the host at sites
where antimicrobials are excluded or are
not active.
 Genetic Origin of Drug Resistance
o Drug-resistant microbes emerge as a
result of genetic change and subsequent
selection processes by antimicrobial drugs.
 Chromosomal Resistance
o Spontaneous mutation in a locus that
controls susceptibility to a given
antimicrobial drug.
o Chromosomal mutants are resistant by
virtue of change in structural receptor for
a drug.
 Extrachromosomal Resistance
o Plasmids –extrachromosomal genetic
elements; carry genes for resistance to
antimicrobial drugs →control the
formation of enzymes capable of
destroying antimicrobial drugs.
Matthew Earl G. Malumay MD-SOON IDCM
o Plasmids determine resistance to
penicillins and cephalosporins by carrying
genes for the formation of β-lactamases.
Cross-Resistance
Microorganisms resistant to a certain drug may
also be resistant to other drugs that share a
mechanism of action, closely related chemically or
have a similar mode of binding or action.
Limitation of Drug Resistance
Ways to Minimize the Emergence of Drug
Resistance:
 Maintaining sufficiently high levels of the drug
in tissues to inhibit original population and
first-step mutants.
 Administering two drugs that do not give
cross-resistance →delays the emergence of
mutants resistant to other drug.
 Avoid exposure of microorganisms to valuable
drug by limiting its use.
CLINICAL IMPLICATIONS OF
DRUG RESISTANCE: Staphylococci
Most staphylococci were susceptible to penicillin
G → β-lactamase producers → resistant to
penicillin G.
Vancomycin –major drug for treatment of MRSA
infections but recovery of isolates with
intermediate resistance and reports of several
cases of high-level resistance to vancomycin have
spurred the search for newer agents.
Newer Agents with Activity against MRSA:
 Daptomycin
 Linezolid
 Quinupristin–Dalfopristin
 Ceftaroline (novel cephalosporin agent)
Sensitive (Staphylococcus aureus):
 Cefuroxime
 Ciprofloxacin
 Clindamycin
 PiperacillinTazobactam
 Ampicillin
 Levofloxacin
 Nitrofurantoin
Resistant (Staphylococcus aureus):
 Nalidixic Acid
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 Antimicrobial Susceptibility Test Diffusion Test Procedures
CLINICAL IMPLICATIONS
OF RUG RESISTANCE:
Gram-Negative Enteric Bacteria
Most drug resistance in enteric bacteria is
attributable to widespread transmission of
resistance plasmids.

Half the strains of Shigella species are now

resistant to multiple drugs.

Salmonellae have also developed resistance to

tetracyclines incorporated into animal feeds.

A clone of Salmonella serotype Typhimurium

phage type DT104 emerged and spread globally
which is resistant to ampicillin, chloramphenicol,
streptomycin, sulfonamides and tetracycline.

Plasmids carrying drug resistance genes occur in

gram-negative bacteria of normal gut microbiota.

Abundant Use of Antimicrobial Drugs →

Suppression of drug-susceptible organisms in gut
microbiota →Favors persistence and growth of
drug-resistant bacteria (Enterobacter, Klebsiella,
Proteus, Pseudomonas and Serratia species and
fungi).

Antimicrobial Activity in Vitro

Antimicrobial Activity in Vitro:
 Potency of Antibacterial Agent in Solution
 Concentration in Body Fluids or Tissues
 Susceptibility of Microorganism to Known
Concentrations of the Drug

Factors Affecting Antimicrobial Activity

 pH of Environment
 Components of Medium
 Stability of Drug
 Size of Inoculum
 Length of Incubation
 Metabolic Activity of Microorganisms

Sensitive (Escherichia coli):

 Cefuroxime

Resistant (Escherichia coli):

 Nalidixic Acid
 Tetracycline
 Clindamycin
 Trimethoprim Sulfamethoxazole

Matthew Earl G. Malumay MD-SOON IDCM 4