AST by Rakesh Prasad Sah

1. Antibiotic Sensitivity Testing
and
Antimicrobial Stewardship
Dr. Rakesh Prasad Sah
Assistant Professor, Microbiology

2. Introduction
• AST (Antibiotic Sensitivity Testing): A test
done to check the effectiveness of a drug against
a bacterium and to select the best drug that acts
against the bacterium.
• The in vitro testing of bacterial cultures with
antibiotics to determine susceptibility of bacteria to
antibiotic therapy.

4. Purposes
• To guide the clinician in selecting the best antibiotic agent for an
individual patient.
• To control the use of in clinical practice.
• To accumulate epidemiological information on the resistance of
microorganisms of public health importance within the community.
• To reveal the changing trends in the local isolates.

5. Types
Qualitative
• For the testing of isolates from “healthy” patients with intact immune
defenses.
• For such as uncomplicated urinary tract infections.
Quantitative
• In the treatment of serious infections such as endocarditis or
osteomyelitis.
• For infections in high-risk patient groups such as
immunocompromised patients (e.g.. Transplant patients).
• Those who are critically ill.

7. To prepare inoculum, take few colonies from primary culture plate with
the help of inoculating loop.
Transfer this to a tube of saline or peptone water.
Incubate at 370C for few hours.

8. Compare the turbidity with 0.5 Mc Farland std. adjust the density by
diluting or by incubating
A lawn culture is inoculated using this broth on Mueller-Hinton
agar
Leave the inoculum to dry for a few minutes at room temperature with the lid
closed.

9. Filter paper discs 6.0 mm in diameter, charged with appropriate
concentration of drugs are placed on a lawn culture
The drug is allowed to diffuse through a solid medium so that a
gradient is established, the concentration being highest near the
site of application
The plate is incubated overnight
The degree of sensitivity is noted by measuring the ‘zone of
inhibition’ of growth around the discs

10. Measurement of diameter
• Using a ruler
▫ on the under-surface of the
plate containing transparent
medium.
• Using a pair of calipers
▫ on the plate containing
opaque medium.

11. Results of KBA
• The zones of inhibition are compared with known values and are
designated as either:
1. Sensitive : respond to treatment
2. Resistant : not to respond to a given antibiotic
3. Intermediate : Treatment at a higher dose

12. Stokes method
A test bacterium is inoculated in the middle third of a plate
The standard strain of a bacterium is inoculated on the upper and lower thirds
of the plate
Standard strains used are Staphylococcus aureus ATCC 25923, E. coli ATCC
25922 and Pseudomonas aeruginosa ATCC 27853

13. Stokes method
Antibiotic discs are applied between the standard and test bacteria
Zones of inhibition around the test and control bacteria are
compared
Advantage: Both control and
test organism is same environment
Disadvantage:
•not accurate and therefore not
used generally
•2 to 4 antibiotics in one plate is
tested
•Laborious

14. Stokes method
• Advantage: Built in controls against many variables and
therefore dependable results
• Disadvantage: not accurate and therefore not used generally

15. Factors affecting the zone of inhibition
1. Diffusibility of drug
2. Disc concentration
3. Nature and composition
of medium and it’s
thickness
Media
Disc
Inoculum
4. pH of medium
5. Time of incubation
6. Turbidity of broth

16. Dilution Methods
• Used to determine the minimal concentration of antibiotic to inhibit
or kill the microorganism.
• Achieved by dilution of antibiotic in either agar or broth media.
MIC
Tube
Dilution
Agar
Dilution

17. Minimum inhibitory concentration
• The lowest concentration of drug that inhibits the
growth of the bacteria isolated from the patient.
Minimum bactericidal concentration
• The lowest concentration of drug that the bacteria
isolated from the patient.

18. Tube Dilution/Broth dilution Method
Isolated organism is added to tubes
containing decreasing amounts of the
antibiotic
Incubation
At 37°C overnight
Lowest concentration of drug that
inhibits growth is the MIC

21. Agar dilution Method
• Serial dilution of the drug are prepared in agar and poured into
plates.
• Many strains can be inoculated on each plate containing an
antibiotic dilutions.
Advantage
• Convenient when several strains are to be tested at the same time.

22. E-Test

23. Epsilometer Test
• Quantitative method of antibiotic sensitivity testing.
• Applies both dilution of antibiotic and diffusion of antibiotic into the
medium.
• Combines the principle of both disk diffusion test and agar dilution
methods.
Diffusion
Dilution
E-Test

24. Procedure
Apply E-strip with known concn of antibiotic
on agar plate inoculated with test organism
Antibiotic diffuses to the agar plate
Incubate & Symmetrical inhibition ellipse is
produced
Antibiotic conc at which ellipse edge
intersects the E-test strip is taken as MIC
value of that antibiotic.

27. Antimicrobial Stewardship
Use of right antimicrobial agents
With right dose
Right route of administration
Right frequency
At right time
To the right patient
Causing least harm to the patient
Antimicrobial
Stewardship

28. Purpose of ASP
• Antimicrobial Resistance (AMR)
▫ Increasing worldwide
▫ MDR (Multidrug resistance)
▫ Extensive use of antimicrobials
• Misuse of Antimicrobials
▫ Over use of it without prescription or
over prescription
▫ Causing development of drug
resistance (MDR) “Super bugs”.

29. Purpose of ASP
• Antimicrobial Usage in Animals
▫ Used in animals for non-therapeutic purpose
(70%) but only 15% for therapeutic purpose.
▫ Over use contribute to resistance  passed to
humans serious infections/ds.
▫ Misuse of antibiotics in farming and
agriculture.
▫ ASP would bring down unnecessary use of
antibiotics.
• Poor Research for Development of
Antimicrobials
▫ Poor research for development of new
antibiotics.

30. Implementation of ASP
• Antimircrobial stewardship committee (ASC) must be
established and polices must be endorsed by the committee.
Important components os ASP are
1. Leadership
2. Antimicrobial stewardship team
3. Infrastructure support
4. Antimicrobial policy
5. Core ASP strategies
6. Education and Training

31. • Leadership
▫ Strong administrative support
▫ Necessary funds and infrastructure support.
• ASP team
▫ Infectious disease physician
▫ Clinical microbiologist or
infection control officer
▫ Clinical pharmacist
▫ Stewardship nurses
▫ Office incharge of pharmacy
Responsible for framing,
Implementation and
monitoring the
compliance of
antimicrobial policy in the
hospital.
Implementation of ASP

32. Implementation of ASP
• Infrastructure Support :
▫ AST (CLSI),
▫ BacT/ALERT,
▫ VITEK

33. Implementation of antimicrobial Stewardship Programme
• Infrastructure Support :
▫ AST (CLSI)
▫ BacT/ALERT
▫ VITEK
▫ Biomarkers
 CRP
 Procalcitonin
▫ Molecular Methods
 PCR
▫ Hospital system including laboratory information 
will boost stewardship programme.
• Antimicrobial Policy

34. Antimicrobial Stewardship
• Core ASP strategies
1. Front end strategies (Formulary restrictions)
 Made available through an approval process (preauthorisation)
 Antimicrobials classifed
 Non-restricted  do not req. approval from ASPT.
 Semi-restricted
 Restricted
Adv
 Impact of this strategy is immidiate
 Most ideal way to achieve AS.
Disadv
 Implemention is not easy
 ASPT approval in emergency situations may not be possible all the
time.
2. Back-end strategies (Prospective audit with intervention
and feedback)
Require approval from AST

35. Antimicrobial Stewardship
2. Back-end strategies (Prospective audit with intervention and
feedback)
• Antimicrobials are reviewed after antimicrobial therapy has been initiated.
(prospective audit with intervention feedback)
• ASP team  stewardship rounddiscuss clinicians  compliance of
antimicrobial policy (dose, renal adjustment, treatment a/c to AST report)
• The clinician will give justification about the non-compliance.
• The prospective audit with intervention and feedback is mutually discussed
between ASP team & clinicians on the cases with follow up.
Advantages
• Widely practiced and most effective.
• Easily accepted by clinicians.
• Impact is sustainable improving the quality of
prescribing antimicrobials.
• Provides opputunity for educating and training
health care personals.
Disadvantages
• More laborious and
intensive
• Takes longer period to
initiate the impact

36. Antimicrobial Stewardship
6. Education and Training

37. Monitoring of Antimicrobial Stewardship
Programme
• Done by
1. Process indicators
▫ It is a policy adherence indicator and can be done by
A. Audit of AS by back-end strategy.
B. Prescription compliance can be checked by
a. Empirical antibiotic given according to infective syndrome.
b. Culture taken before the start of antibiotics.
c. Modification of the empirical antibiotics a/c AST report.
d. Surgical antimicrobial prophylaxis a/c formulated policy.
C. Administrative compliance include
a. Correct administration of antibiotic (dose, frequency & route).
b. Correct administration of surgical antimicrobial prophylaxis.
Process
Indicators
Outcome
Indicators

38. Antimicrobial Stewardship
2. Outcome indicators
A. Antimicrobial usage
a) Defined daily dosage (DDD)
b) Days of therapy (DOT)
B. Antimicrobial resistance (AMR)
 Analysed by AMR surviellance conducted periodically.
C. Clinical
a) Morbidity: length of patient stay in hospital.
b) Mortality : deaths due to infectious ds.
D. Financial outcome indicator
 Can be analysed by calculating the cost of antimicrobial
per patient day or per year or per admission.

39. Rational use of antimicrobials
• Can be done by considering the following
conditions.
1. Prescribe Antibiotics Rationally
2. Antimicrobial Therapy according to site of infection
3. Correct administration of Antimicrobials
4. Rational use of Empirical Therapy
5. Antimicrobial Therapy in some specific conditions
6. Drug monitoring
7. Stoppage of Antimicrobial Therapy Appropriately

40. Antimicrobial Stewardship
1. Prescribe Antibiotics Rationally
▫ Viral Infections : Do not use.
▫ Diarrhoea : Oral rehydration therapy
▫ Prophylaxis: do not use antibiotic routinely to
prevent infections except in some situations e.g.
cotrimoxazole in HIV/AIDS infected patients.
2. Antimicrobial Therapy according to site of infection e.g.
UTI.
3. Correct administration of Antimicrobials
Right antimicrobial
right dosage
right frequency
right duration

41. • Rational use of Empirical Therapy
▫ Empirical antibiotic should be given a/c the infective
syndrome and it’s causative agent.
▫ Emiprical therapy  modified subsequently AST
report
• Drug monitoring
▫ Therapeutic efficacy of antimicrobial agent  in-vivo
activity related to
 Pharmacokinetic
 Pharmacodynamic
▫ E.g. Amikacin & Vancomycin  concn in serum 
higher than MIC value.
Antimicrobial agents
not on in-vitro
susceptibility test
(MIC)

42. Antimicrobial Stewardship
• Stoppage of Antimicrobial Therapy Appropriately
▫ Should be stopped at appropriate time  on clinical
improvement/negative culture report or biomarkers of
bacterial infection.

43. Antimicrobial Stewardship

44. Antimicrobial Stewardship