IA. General microbiology | 12. Antibiotic sensitivity tests in laboratory. Chemoprophylaxis. Antibacterial therapy.

Question 1

I. Definitions
1. What is antibiotic?

Answer 1

natural agent synthesized by a bacterial or fungal strain that can inhibit or kill other microorganisms

Question 2

I. Definitions
2. What is synthetic antibiotics?

Answer 2

a naturally synthetized agent which is modified chemically in order to have antimicrobial + pharmacokinetic properties advantages

Question 3

I. Definitions
3. What is a Chemotherapeutic agent?

Answer 3

an antimicrobial agent synthesized chemically

Question 4

I. Definitions
4A. What is a Bacteriostatic effect?

Answer 4

The antimicrobial agent (AA) can only inhibit the growth of the microorganisms but is unable to kill them within a short period of time and rely on host defense mechanisms for final eradication of the infection
- This is determined in vitro by testing standardized concentrations of organisms against a series of antimicrobial dilutions
- MIC (minimum inhibitory concentration): lowest concentration that inhibits the growth of the organisms after overnight incubation

Question 5

I. Definitions
4B. How is Bacteriostatic effect determined?

Answer 5

This is determined in vitro by testing standardized concentrations of organisms against a series of antimicrobial dilutions

Question 6

I. Definitions
4C. What is MIC(minimum inhibitory concentration)? (Bacteriostatic effect)

Answer 6

lowest concentration that inhibits the growth of the organisms after overnight incubation

Question 7

I. Definitions
5A. What is Bactericidal effect?

Answer 7

The antimicrobial agent (AA) can kill growing microorganisms within a short period of time
- This is determined in vitro by exposing a standardized concentration of organisms to a series of antimicrobial dilutions
- MBC(minimum bactericidal concentration): lowest concentration that kills 99.9% of the population

Question 8

I. Definitions
5B. How is Bactericidal effect determined?

Answer 8

The antimicrobial agent (AA) can kill growing microorganisms within a short period of time
- This is determined in vitro by exposing a standardized concentration of organisms to a series of antimicrobial dilutions

Question 9

I. Definitions
5C. What is MBC (minimum bactericidal concentration)? (Bactericidal effect)

Answer 9

MBC (minimum bactericidal concentration): lowest concentration that kills 99.9% of the population

Question 10

I. Definitions
6. What is Selective toxicity?

Answer 10

• The antibiotic has an effect only on the bacteria, but not on the human host.
• The target of the antibiotic (cell wall, ribosome) only present in the bacteria but not in the human host→ only toxic for microbe

Question 11

I. Definitions
7A. What is Chemotherapeutic Index (Ehrlich)?

Answer 11

• Def.: The highest concentration of the antimicrobial agent that can be tolerated by the host organism divided by the lowest concentration of the antimicrobial agents that inhibit or kill the microorganism
• Chemotherapeutic Index (CI) = Dosis tolerata maxima (DTM)/Dosis curativa maxima (DC
  +) When the drug is safer (e.g. B-lactams)
  +) When the l → the drug is more toxic (e.g. glycopeptide, aminoglycoside)

Question 12

I. Definitions
7B. What are the features of calculating with Chemotherapeutic Index (Ehrlich)?

Answer 12

Chemotherapeutic Index (CI) = Dosis tolerata maxima (DTM)/Dosis curativa maxima (DCM)
- When the drug is safer (e.g. B-lactams)
- When the l → the drug is more toxic (e.g. glycopeptide, aminoglycoside)

Question 13

I. Definitions
8A. What is Antibiotic susceptibility testing?

Answer 13

An Antibiogram is the result of an antibiotic sensitivity test; it’s an in vitro sensitivity, but the correlation of in vitro to in vivo sensitivity is often high enough for the test to be clinically useful

Question 14

II. Antibiotic susceptibility testing
1. What are the methods of Antibiotic susceptibility testing?

Answer 14

Question 15

II. Antibiotic susceptibility testing - Dilution methods
2. What are the 4 dilution method that we can use for Antibiotic susceptibility testing?

Answer 15

1. Broth dilution methods (tube and microdilution)
2. Tube dilution method
3. Microdilution method
4. Agar dilution method

Question 16

II. Antibiotic susceptibility testing - Dilution methods
3. What are the 5 steps of Broth dilution methods (tube and microdilution)?

Answer 16

1) We use sterile Muller-Hinton broth

2) Make 2-fold dilution of antibiotic in broth (eg 256 μg/ml, 128 μg/ml, 64 μg/ml, 32 μg/ml, 16 μg/ml, 8 μg/ml, ….)

3) Add culture of the test organisms to the prepared dilutions

4) Incubation: after adding test organism we incubate the tubes or the microtiter plate (37 deg C for
24 hour)

5) Then determine MIC of antibiotic (in μg/ml)

Question 17

II. Antibiotic susceptibility testing - Dilution methods
4. What are the features of Tube dilution method?

Answer 17

• We use test tubes for this test
• Use about 1ml total broth volume in which we make 2-fold dilution of antibiotic
• The lowest concentration (highest dilution) of test agent preventing appearance of turbidity (growth) is considered to be the MIC

Question 18

II. Antibiotic susceptibility testing - Dilution methods
5. What are the features of Microdilution method?

Answer 18

• Broth microdilution method is internationally accepted susceptibility testing method
• Here, broth volume for each microbial agent is 0.1 ml
• It is performed in a 96 well microtiter plate

Question 19

II. Antibiotic susceptibility testing - Dilution methods
6A. What are the features of Agar dilution method?

Answer 19

• Serial dilution of antibiotics are prepared in agar and poured into Petir dish
• Inoculation of the plates with the bacterial strains which we want to test (a control plate also necessary)
• One concentration of antibiotic/petri dish
  +) Possible to test several different strains/Petri dish
• Interpretation: examine the drug-free control growth of the test organisms for viability and purity and place the plates on a dark background and examine them for the lowest concentration that inhibits visible growth

Question 20

II. Antibiotic susceptibility testing - Dilution methods
6B. What is the Interpretation for Agar dilution method?

Answer 20

Interpretation: examine the drug-free control growth of the test organisms for viability and purity and place the plates on a dark background and examine them for the lowest concentration that inhibits visible growth

Question 21

II. Antibiotic susceptibility testing - DIFFUSION methods
7. What are the 3 major diffusion methods?

Answer 21

1. Kirby-Bauer disc diffusion method:
   - commonly used for routine testing
   - antibiotic susceptibility test in determining what choice of antibiotics should be used when treating an infection
2. Punching diffusion method
3. E-test: determines antimicrobial sensitivity

Question 22

II. Antibiotic susceptibility testing - DIFFUSION methods
8A. What is Kirby-Bauer disc diffusion method?

Answer 22

• Antibiotic susceptibility test in determining what choice of antibiotics should be used when treating an infection
• Commonly used for routine testing

Question 23

II. Antibiotic susceptibility testing - DIFFUSION methods
8B. What are the 4 steps of Kirby-Bauer disc diffusion method?

Answer 23

1. Prepare the lawn culture of the isolate by streaking the swab all over the surface of the medium while rotating the plate
2. Leave the inoculum to dry at room temp with lid closed
3. The antibiotic disks can be placed on the inoculated plates using a pair of sterile forceps (MAX 6 disks can be placed
4. After overnight incubation, measure diameter of each inhibition zone and record in mm

Question 24

II. Antibiotic susceptibility testing - DIFFUSION methods
8B. In Kirby-Bauer disc diffusion method, step 3: “The antibiotic disks cay be placed on the inoculated plates using a pair of sterile forceps (MAX 6 disks can be placed”
=> What do we need to take into account?

Answer 24

• Spaced evenly, approx 15mm from edge of the plate
• Do NOT move disc once in contact with the agar surface since some of the compound
  diffuses almost instantaneously
• Antibiotic diffuses from disc into again in decreasing amounts the further it is away from the disc
• If organism is killed or inhibited by the concentration of the antibiotic, there will be NO
  growth in the immediate area around the disc (ZONE OF INHIBITION)

Question 25

II. Antibiotic susceptibility testing - DIFFUSION methods
9. What are the features of Punching diffusion method?

Answer 25

• Similar to disc diffusion method, but we make antibiotic solution ourselves
• Punch the holes into the agar for these solutions

Question 26

II. Antibiotic susceptibility testing - DIFFUSION methods
10. What are the features and steps in E-test?

Answer 26

E-test: determines antimicrobial sensitivity
- Plastic strips impregnated with decreasing antibiotic concentrations
- Consists of predefined gradient of antibiotic concentration
- Strips applied on agar microbial culture and incubated
- Elliptic inhibition zone intersects the MIC value scale at the level of MIC

Question 27

III. Antibacterial Therapy
1. What are the 2 types of Antibacterial Therapy?

Answer 27

1. Targeted therapy
2. Empirical therapy

Question 28

III. Antibacterial Therapy
2. What is targeted therapy?

Answer 28

When bacteria is already known and we can perform an antibiotic susceptibility testing test (antibiogram)

**The spectrum of the antibiotics should be the most narrow one

Question 29

III. Antibacterial Therapy
3. What are the features of Empirical therapy?

Answer 29

When bacteria is not known (yet) so we use broad spectrum antibiotics to act against all possible causative agents
- Depending on local resistance data
- Sampling for cultivation before the start of treatment
- After identification→ changing to targeted treatment

**The spectrum of the antibiotics should be the most narrow one

Question 30

III. Antibacterial Therapy
4. What are the 5 factors that we need to consider in Antibacterial Therapy?

Answer 30

1. Side effects
2. For how long?
3. Dose
4. Pharmacokinetics
5. Cid/static agent?

Question 31

III. Antibacterial Therapy
5. Why do we need to consider Side effects in Antibacterial Therapy?

Answer 31

• Toxicity: direct organ or cell damage
• Allergy: hypersensitivity, anaphylaxis
• Toxins produced by the microbes
• Disbacteriosis: destruction of the normal flora

Question 32

III. Antibacterial Therapy
6. Why do we need to consider “For how long?” in Antibacterial Therapy?

Answer 32

• Acute infection: 5 days min
• Severe infection: 8-14 days
• Endocarditis/Sepsis: 4-6 weeks (tbc:8-12 months)

Question 33

III. Antibacterial Therapy
7. Why do we need to consider Dose in Antibacterial Therapy?

Answer 33

• Age, weight, body surface, liver and kidney function

Question 34

III. Antibacterial Therapy
8. Why do we need to consider Pharmacokinetics in Antibacterial Therapy?

Answer 34

• Absorption– p.o., iv
• Elimination– kidney, liver
• Tissue diffusion– blood-brain barrier, gallbladder, urine

Question 35

III. Antibacterial Therapy
9. Why do we need to consider Cid/static agent in Antibacterial Therapy?

Answer 35

Cid/static agent?
- Bacteriostatic only can be used when patient is immunocompetent