Antimicrobial susceptibility testing

& antimicrobial agents

Jose G. Perez, Jr., RMT, MSMT

Licensed Professional Teacher
Occupational & Safety Health Officer
TERMINOLOGIES

Chemotherapy is the use of chemical substances (drugs) to treat

disease

Selective toxicity - property of the antibiotic in which it should be

toxic to the bacteria and not to the host

Side effects - Symptoms of host poisoning

 TERMINOLOGIES
Antimicrobial drugs which has the capacity to inhibit the growth
or kill microbes in dilute solution. drugs used for treating infectious
diseases (caused by microbes)
Preparation -Antibiotic, semi-synthetics , synthetics
Action - Antibacterial, antifungal, antiviral, antiprotozoal
Antibiotics naturally produced by microbes
Synthetics derived from naturally occuring antimicrobials & are
synthesis in the laboratory
TERMINOLOGIES
Narrow spectrum - can kill or inhibit the growth of gram-positive OR
gram-negative bacteria but not both

Broad spectrum - can kill or inhibit the growth of gram-positive AND

gram-negative bacteria

Bactericidal - kills bacteria

Bacteriostatic - inhibits the multiplication of bacteria but DOES NOT

kill it
TERMINOLOGIES
MIC (Minimum Inhibitory Concentration) - the lowest concentration of
the drug that prevents the growth of bacteria

MBC (Minimum Bactericidal Concentration) - the lowest concentration

of the drug that kills 99.9% of the organism
TERMINOLOGIES
Drug combination

Addition - the action of two drugs is equal to their sum

1+1=2

Synergism - 2 drugs - combined but the action is better than the sum
1+1=3

Antagonism - one drug decreases the activity of the other

1+1=1
Requirements of AST
bacteria subjected to AST - isolated in pure culture from - sample,

standard reference methods - used for identification so bacteria are

consistently & correctly identified to the genus &/or species level,

bacterial isolates - most important & other isolates - stored for future
analysis (either lyophilisation or cryogenic preservation at 70C to
80C).
FACTORS INFLUENCING AST - DETERMINED, OPTIMISED, & DOCUMENTED - SOP

Isolated Bacteria or other organisms (pure culture) used in AST testing

should be from a fresh culture,

Agar or broth composition & preparation (e.g. pH, cations, thymidine or

thymine use of supplemented media).

the content of antimicrobial in the carrier (antibiotics used in microtitre

plates, disk, strip, tablet)
FACTORS INFLUENCING AST - DETERMINED, OPTIMISED, & DOCUMENTED - SOP

composition of solvents and diluents for preparation of

antimicrobial stock solutions,
growth and incubation conditions (time, temperature, atmosphere
e.g. CO
agar depth,
number of concentrations tested per broth & agar dilution,
test controls to be used, including the reference organisms used,
subsequent interpretive criteria (clinical breakpoints,
epidemiological cut-off values).
 The selection of an AST methodology may be based on
the following factors:

ease of performance,
flexibility,
adaptability to automated or semi-automated systems,
cost,
reproducibility,
reliability,
accuracy,
the organisms and the antimicrobials of interest in that
particular OIE Member,
availability of suitable validation data for the range of
organisms to be susceptibility tested.
 Disc Diffusion
standard AST?
inoculum is swabbed onto MHA,
& paper disks impregnated with
antimicrobial are placed on the
agar.
overnight incubation diameter
(ZOI) around each disk is
measured.
Based on ZOI a qualitative
report of S, I, R is determined.
Kirby Bauer
Medium: Mueller-Hinton Agar
Depth of Medium: 4 mm
pH: 7.2-7.4
Distance from each disk: 30 mm from disk
center
Distance from the edge of plate: 10 mm
Uses 0.5 McFarland Turbidity Standard
(1.5 x 108 CFU/mL)
Components: 99.5 mL of 1% H2SO4 and 0.5 mL
1.175% BaCl2
 Agar Well Diffusion
inoculum is swabbed onto MHA,
& agar well is made using cork
borer, then antimicrobial are
placed into the wells.
overnight incubation diameter
(ZOI) around each well is
measured.
Based on ZOI a qualitative
report of S, I, R is determined.
Minimum Inhibitory Concentration (MIC) Test

Figure 10.10
E-Test(MIC)

Figure 10.11
COMPLEXITY OF ANTIMICROBIAL THERAPY
Antimicrobial (=antibiotic or synthetic drug)

Antimicrobial Administration
Wrong Drug Drug Delivery
Wrong Spectrum Problems
of Activity (informed Oral (destruction/poor
best guess), uptake), Intravenous
Few Targets on or Intramuscular
Pathogen (inconvenient),
(fungi, protozoa, Topical, Poor Tissue
viruses) Uptake, Injection into
Body Cavity
Side Effects
Toxicity to Host,
Allergic Reaction, Drug Retention
Normal Flora Problems
Disruption Elimination from Host,
(superinfection) Inactivation by Host
Selective Toxicity & Successful Delivery
COMPLEXITY OF ANTIMICROBIAL THERAPY
Selective Toxicity & Successful Delivery
Toxins
Exotoxins,
Development of
Endotoxins
Resistance
(in Gram-negative
Evasion,
septicemia
Mutation-Mediated,
antibiotic treatment
Acquired (R plasmids)
can make situation
even worse)

Prevent Resistance Prevention of

by Using Growth
Only as Necessary, Bactericidal,
At sufficiently high Bacteriostatic,
doses for sufficiently Host Defenses
long periods, In (elimination of
Combination pathogen from body)