Microbiological culture sensitivity test. antimicrobial sensitivity test

1. Microbiological Culture Sensitivity Test.
Antibiotic Sensitivity Test ( AST )
Akhil Joseph
Pharm.D IVth Year.
Reg.No: 13Q0402

2.  According to the new ISO 20776-1 standard, which is
valid all over the world, these terms are defined as
follows:
 Susceptible (s): A bacterial strain is said to be
susceptible to a given antibiotic when it is inhibited in
vitro by a concentration of this drug that is associated
with a high likelihood of therapeutic success.
 Intermediate (i): The sensitivity of a bacterial strain
to a given antibiotic is said to be intermediate when it
is inhibited in vitro by a concentration of this drug that
is associated with an uncertain therapeutic effect.
 Resistant (r): A bacterial strain is said to be resistant
to a given antibiotic when it is inhibited in vitro by a
concentration of this drug that is associated with a high
likelihood of therapeutic failure.

3. mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L
Ampicillin 0.25 0.5 1 2 4 8 16 32
Ampicillin/sulb
actam
0.25 0.5 1 2 4 8 16 32
Piperacillin/taz
obactam
0.5/2 1 2 4 8 16 32 64
Cefuroxime 0.125 0.25 0.5 1 2 4 8 16
Cefotaxime 0.125 0.25 0.5 1 2 4 8 16
Imipenem 0.125 0.25 0.5 1 2 4 8 16
Gentamicin 0.125 0.25 0.5 1 2 4 8 16
Doxycyclin 0.125 0.25 0.5 1 2 4 8 GC
Cotrimoxazole 1 2 4 8 16 32 64 128
Ciprofloxacin 0.03 0.06 0.125 0.25 0.5 1 2 4
Levofloxacin 0.03 0.06 0.125 0.25 0.5 1 2 4
Moxifloxacin 0.03 0.06 0.125 0.25 0.5 1 2 4
Schematic representation of a microtitration plate for the determination of
minimal inhibitory concentrations (MICs)

4.  The results for a particular tested strain of E. coli.
 The MIC values for this strain and the sensitivity ratings that will
be assigned to them henceforward by EUCAST (The European
Committee on Antimicrobial Susceptibility Testing) , are:
 ampicillin = 4 mg/L (i); ampicillin/sulbactam = 1 mg/L
(i); piperacilin/tazobactam = 2 mg/L (s); cefuroxime = 4
mg/L (i); cefotaxime = 0.125 mg/L (s); imipenem = 0.5 mg/L
(s); gentamicin = 0.25 mg/L (s); doxycycline = 8 mg/L
(Ø); cotrimoxazole >128 mg/L (r); ciprofloxacin <0.03 mg/L
(s); levofloxacin <0.03 mg/L (s); moxifloxacin = 0.06 mg/L
(s);
 GC, growth control; Ø, no data, because the combination of
organism and antibiotic is unsuitable; i, intermediate; s,
susceptible; r, resistant.

5. INTRODUCTION
 Antimicrobial susceptibility testing (AST) is indicated
for pathogens contributing to an infectious process that
warrants antimicrobial therapy if susceptibility to
antimicrobials cannot be predicted reliably based on
knowledge of their identity.
 Some organisms have predictable susceptibility to
antimicrobial agents (ie, Streptococcus pyogenes to
penicillin), and empirical therapy for these organisms is
typically used. Therefore, AST for such pathogens is
seldom required or performed. In addition, AST is
valuable in evaluating the activity of new and
experimental compounds and investigating the
epidemiology of antimicrobial resistant pathogens.

6.  Once we have identified the bacterium which is causing
the infection we need to find out the antibiotics that
would be effective against it. This is done by antibiotic
sensitivity testing.
 Antibiotic sensitivity or microbiological culture
sensitivity are the in-vitro procedures to determine
the susceptibility of bacteria to antibiotics.
 Because susceptibility can vary even within
a species (with some strains being more resistant than
others), antibiotic susceptibility testing (AST) is
usually carried out to determine which antibiotic will be
most successful in treating a bacterial infection in vivo.

7.  The same can be used to study the emergence of
antibiotic resistance and spread of resistant organism
in a population.
 Antibiotic sensitivity testing will control the use of
Antibiotics in clinical use.
 Testing will assist the clinicians in the choice of drugs
for the treatment of infections.

8. Why is sensitivity analysis done ?
 Usually almost all the bacteria in infectious disease are drug
resistant.
 Unfortunately, nowadays many bacteria are resistant to
common antibiotics.
 Thus sensitivity tests are better tool to quickly determine if
bacteria are resistant to certain drugs.
 Hence sensitivity test is performed to select the correct
antimicrobial drug of choice, hence cost effective and
rational therapy can be provided.
 Lower the risk of emergence of antibiotic resistance.
 It may also help to identify the pathogen.

9. Limitations
o It helps us to measure only the antimicrobial activity
against a bacteria under laboratory conditions and not
in the patients.
o The patients clinical condition, type and site of
infection, drug hypersensitivity, ADME, characters of
the patients are not taken in to consideration in
sensitivity testing techniques.

10. Culturing and Sensitivity Testing
Specimen Collection.
Samples must be collected and handled properly to obtain reliable
results.
Poor collection techniques may result in lack of bacterial growth or
abundant growth of bacterial contaminats.
Sample shoud be collected before the antibiotic therapy to assure
the best growth of the pathogen.
If antibiotic therapy is already initiated, then the sample must be
collected before the next dose.

11. Identifying Bacteria
 When a sample is submitted to laboratory for bacterial culture and
antibiotic sensitivity, the clinician should include the information
like the site of sample collection and type of lesion. This help the
microbiologist to choose which nutrient media and growth
conditions to be used.
 Samples of bacterial culture are applied to plates of various growth
media with a sterile loop, effectively spreading the bacterial
organism over the surface of each plate in a single layer.
 Once inoculated the plates are incubated in an environment with
controlled temperature, humidity, oxygen and carbon dioxide
levels are optimum for bacterial replication.
 Each bacterial organism grows into a cluster called a colony, and
individual colonies are inoculated onto new separate media,
creating pure samples.
 Identification of the cultured bacteria is based on the
characteristics of colony growth and appearance as well as
biochemical testing of the individual colonies.

12. Antibiotic Sensitivity Testing.
 Once identified the bacteria undergoes
testing to identify the antibiotic most
likely to inhibit their growth.
 For this purpose generally two methods
are used,
1) Disk Diffusion techniques.
2) Broth Dilution techniques.

13. Disk Diffusion techniques.
Agar Disk Diffusion Testing
 In many clinical microbiology laboratories an agar disk
diffusion method is routinely used for the testing of
common, rapidly growing, and some fastidious
bacterial pathogens, allowing categorization of most
such isolates as susceptible, intermediate, or resistant
to a wide range of antimicrobial agents.
 A disc of blotting paper is impregnated with a known
volume and appropriate concentration of antimicrobial
placed on a plate of sensitivity agar inoculated with
test organism.

15.  The antimicrobial agents then diffuse from the disks
through the agar, and as the distance from the disks
increases, the drug concentrations decrease in a
logarithmic fashion, creating gradients of drug
concentrations in the medium around the disks.
 Simultaneously with the diffusion of the drugs, the
bacteria inoculated to the agar surface not inhibited
by the concentrations of the antibiotics in the agar
multiply, creating a visible lawn of growth. In areas
where the test organism is inhibited by the
antimicrobial agents, growth fails to occur, resulting in
zones of inhibition around each active drug. The
inhibitory zone diameters are influenced by the
diffusion rates of the various antimicrobial agents
through the agar
 After 24 hours, the culture is examined for areas of
growth around the disc.

18.  The zone sizes are inversely proportional to the logarithms of the
antibiotic MICs. After incubation at recommended temperatures,
atmospheric conditions, and times, depending on the pathogen under
study, the diameters of the zones of inhibition are measured in
millimeters and compared to a standard table of predetermined zone
widths representing antibiotic concentrations in the agar that
correlates with that of the antibiotic concentration achievable in the
plasma of a patient using the manufacturers recommended dosage.
 If the zone of inhibition is wider than the pre determined zone, the
bacterial species is considered to be susceptible(S) to the antibiotic. If
bacteria grows within the pre determined zone width the species is
considered as resistant (R). An intermediate (I) designation is used if
the zone of inhibition approximates the predetermined zone width.
 Growth for sensitive strains are inhibited for a distance while for
resistant strains it grows up to the edge of the disc.
 The volume, moisture content, PH, constituent of agar medium,
concentration, storage and application of dose influence the diffusion
technique.
 Agar diffusion sensitivity tests are carried out either by Kirby-Bawer
(KB) method, ICS method or by Stocks method.
 Modified KB method is recommended by the National Committee for
clinical Laboratory Standards (NCCLS) and the WHO.

19. ADVANTAGES
 (1) it is technically easy to perform and results are reproducible,
 (2) the reagents and supplies are inexpensive,
 (3) it does not require the use of expensive equipment,
 (4) it generates categorical interpretive results well understood by
clinicians, and
 (5) it allows for considerable flexibility in the selection of
antibiotics for testing.
DISADVANTAGES
 The exact conc. of antibiotic that inhibited the bacterial growth is
not known.

20. Broth Dilution techniques.
 Dilution sensitivity tests usually measures the minimum
inhibitory concentration (MIC) or minimum bactericidal
concentration (MBC) required to kill the bacteria.
 Test tubes or wells containing increasing concentrations of each
antibiotic to be tested, from 0.0312 to 512mcg/ml, are inoculated
with a fixed volume of nutrient broth containing a standard
concentration of bacteria is added.
 The conc. Of antibiotic in each tube is double as that of previous
tube.
 After overnight incubation the tubes are checked for turbidity,
turbidity indicates growth of bacteria, the lowest antimicrobial
required to prevent visible growth is taken in to consideration.
 MIC is the lowest concentration of antibiotic that inhibits the
bacterial growth.

23.  Dilution technique needs,
 Careful standardization
 Broth and agar medium
 Antimicrobial solution
 Incubation time and
 Dilution time

24. Ideally, clinicians should always choose a drug to which
the identified bacteria are considered susceptible and
should avoid agents to which they are intermediate or
resistant.

25. General requirements for sensitivity
testing.
1. Sensitivity testing agar.
Suitable media include Mueller Hinton agar, Iso sensitest agar
and Gibco sensitivity testing agar no.2.
Mueller Hinton agar(MHA)
Composition
 Meat infusion 2.0 g/l
 Casein hydrolysate 17.5 g/l
 Starch 1.5 g/l
 Agar-agar 13.0 g/l

26. 2.ANTIMICROBIAL DISC
 This disc should be refrigerated at a temperature
instructed by the manufacturer.
 This should not be used after expiry date.
 The working stock disc should be warmed to room
temperature, avoid keeping in direct sunlight.

27. ANTIMICROBIAL RESISTANCE
 Antimicrobial resistance can arise in bacteria in several
ways.
 Microbes acquire resistance after a change in their
DNA.
 Such changes may occur by
 genetic mutation i.e. by alteration in the structure of
their own DNA.
• genetic exchange i.e. by acquisition of extra-
chromosomal DNA from other bacteria.

28.  Genetic exchange is the most common cause of
serious clinical drug resistance because it can produce
resistance to multiple drugs.
 In genetic exchange , the resistance genes are
transferred from one bacterial species to another by
means of discrete, movable, extra chromosomal DNA
elements called TRANSPOSONS.

29.  Transfer of transposons between bacteria can occur by
• Conjugation i.e, direct physical mating between
bacteria.
• Transduction i.e, through the agency of
bacteriophages.
• Transposition i.e, by means of plasmids which are
transferable, extra chromosomal DNA molecule.

30. DRUG RESISTANCE
It refers to unresponsiveness of a micro-
organism to an antimicrobial agent.
They are of 3 types:
i. Natural resistance
ii. Acquired resistance
iii. Cross resistance.

31. NATURAL RESISTANCE
 Some microbes have always been resistant to certain
AMA.
 They lack the metabolic process or the target site
which is affected by the particular drug.
 eg. gram negative bacilli are normally unaffected by
Pencillin G
 M.tuberculosis is insensitive to tetracyclines.

32. ACQUIRED RESISTANCE
 It is the development of resistance by an organism
(which was sensitive before) due to the use of an AMA
over a period of time.
 Eg; methicillin resistant Staphylococcus aureus.

33. CROSS RESISTANCE
 Cross-resistance is the tolerance to a usually toxic
substance as a result of exposure to a similarly acting
substance.
 It is a phenomenon affecting e.g. pesticides and
antibiotics as an example.