بكتريا عملي

1. Antimicrobial Susceptibility Testing
By
Dr. Nabil El Aila
Assistant Professor of Molecular Microbiology
Medical Technology Department
Al -Aqsa University

2. Introduction
Antibiotics = a natural substance produced by a micro-organism to kill another
•They include the culture extracts and filtrates of fungi such as penicillium and cephalosporium and bacteria such as streptomyces and bacillus species.
Antiinfectives/Anti-microbrial = any agent (natural or synthetic) that kills pathogens (microbes)
•They include the sulfonamides, trimethoprim, cotrimoxazole, nitrofurantoin, nalidixic acid, metroniadazole, P. aminosalicylic acid, isoniazid and ethambutol.
•Chemical Antifungal agents include nystatin, and flucytosine.
Dr. Nabil El Aila General Microbiology

4. Mechanism of Action of Antibiotics
•Inhibition of Cell Wall Synthesis
•Disruption of Cell Membrane
•Inhibition of Protein Synthesis
•Interference with Metabolic Processes
Dr. Nabil El Aila
General Microbiology

5. Classification:
Cell wall synthesis inhibitors
•Beta-lactams (penicillins, cephalosporins, aztreonam, imipenem)
•Poly-peptides (bacitracin, vancomycin)
Protein synthesis inhibitors
•Aminoglycosides
•Tetracyclins
•Macrolides
•Chloramphenicol
•Clindamycin
Folate antagonists
•Sulfonamides
•Trimethoprim
•Quinolones

6. Classification
Beta-lactams
Poly-peptides
Cell wall synthesis inhibitors
Penicillins
Cephalosporins
Monobactam (Aztreonam)
Carabpenems (Meropenem/Imipenem)
Bacitracin
Vancomycin

7. Cell wall synthesis inhibitors
Penicillins
Cephalosporins
Beta lactams
Narrow spectrum – penicillinase sensitive
Benzylpenicillin, Phenoxymethylpenicillin
Narrow spectrum – penicillinase resistant
Methicillin, Oxacillin, Cloxacillin, Dicloxacillin
Broad spectrum penicillins
Ampicillin, Amoxicillin
Extended spectrum penicillins
Carbenicillin, Ticarcillin, Mezlocillin, Pipercillin
First generation –
β-lactamase sensitive
Cephazolin, Cephalexin
Third generation – mostly β-lactamase resistant
Cefotaxime, Ceftriaxone
Second generation – β- lactamase sensitive
Cefaclor, Cefamanodole
Cefoxitin
Fourth generation – mostly β-lactamase restistant
Cefepime, Cefpirome

8. β-Lactams
Dr. Nabil El Aila
General Microbiology

9. How do they work?
1.The β-lactam binds to Penicillin Binding Protein (PBP)
2.PBP is unable to crosslink peptidoglycan chains
3.The bacteria is unable to synthesize a stable cell wall
4.The bacteria is lysed

10. Classification:
Cell wall synthesis inhibitors
•Beta-lactams (penicillins, cephalosporins, aztreonam, imipenem)
•Poly-peptides (bacitracin, vancomycin)
Protein synthesis inhibitors
•Aminoglycosides
•Tetracyclins
•Macrolides
•Chloramphenicol
•Clindamycin
Folate antagonists
•Sulfonamides
•Trimethoprim
•Quinolones
Dr. Nabil El Aila
General Microbiology

11. Classification
Aminoglycosides
Protein synthesis inhibitors
Tetracyclines
Macrolides
Clindamycin
Chloramphenicol

12. Classification
Aminoglycosides
Protein synthesis inhibitors
• Gentamicin
• Tobramycin
• Streptomycin
• Neomycin
• Kanamycin
• Amikacin
Tetracyclines
Macrolides
• Tetracycline
• Doxytetracycline
• Minocycline
• Doxycycline
• Erythromycin
• Azithromycin
• Clarithromycin

13. Classification:
Cell wall synthesis inhibitors
•Beta-lactams (penicillins, cephalosporins, aztreonam, imipenem)
•Poly-peptides (bacitracin, vancomycin)
Protein synthesis inhibitors
•Aminoglycosides
•Tetracyclins
•Macrolides
•Chloramphenicol
•Clindamycin
Folate antagonists
•Sulfonamides
•Trimethoprim
•Quinolones
Dr. Nabil El Aila
General Microbiology

14. Classification
Sulfonamide
Folate antagonist
• Sulfadiazine
• Sulfadimidine
• Sulfamethoxazole
Quinolones
Trimethoprim
• Nalidixic acid
• Ciprofloxacin
• Levofloxacin
• Ofloxacin
• Norfloxacin
• Travofloxacin

16. Resistance to Antibiotics
•By genetic mutation which changes the proteins and other components of bacterial cells which Antimicrobial use as binding sites.
• Gene transfer: plasmids (via conjugation and transduction); transposons
•By bacteria producing enzymes (β-lactamase) that destroy or inactivate Antimicrobial.
•By bacteria changing to other metabolic systems not affected by the Antimicrobial being used.
•Microbes may cease active uptake of certain drugs (tetracyclines)
Dr. Nabil El Aila
General Microbiology

17. Resistance to Antibiotics
•Changes in receptors which decrease antibiotic binding and action
•Microbes may synthesize compounds that antagonize drug actions
•By bacteria altering the permeability of their cell membrane making it difficult for Antimicrobial to enter.
•Antibiotic use promotes the emergence of drug-resistant microbes
(especially the use of broad-spectrum antibiotics)
•!!! The more ABs are used, the greater the chance of resistance
•
Dr. Nabil El Aila
General Microbiology

18. Dr. Nabil El Aila
General Microbiology

19. Resistance avoided/delayed by
•Using antibiotics only when absolutely needed and indicated:
•Antibiotics often abused for viral infections (diarrhea, flu- symptoms, etc.)
•Starting with narrow-spectrum drugs
•Limiting use of newer drugs
(Minimizing giving antibiotics to livestock)
•Identifying the infecting organism
•Defining the drug sensitivity of the infecting organism
•Considering all host factors:
•site of infection, inability of drug of choice to penetrate the site of infection, etc.
– Using AB combinations only when indicated:
•Severe or mixed infections, prevention of resistance (tuberculosis)
•Worldwide more than 500 metric tons antibiotics are used anually !!!

20. Routine Susceptibility Tests
•Disk diffusion (Kirby Bauer)
•Etest

21. Disk diffusion Method
 This method, commonly referred to as the Kirby-Bauer test, provides a qualitative measure of the ability of an antimicrobic to inhibit the growth of a rapidly growing bacterium.

22. Disk diffusion Method
 Disks containing a given concentration of an antimicrobic
are placed on a confluently inoculated agar plate and
incubated for 16 to 24 hours.
 At the end of the incubation period, zones of growth
inhibition are measured across the disk diameter and
recorded to the nearest millimeter.

23. Disk Diffusion Method
Procedure of the Kirby-Bauer test
1、Growth Media
2、Bacteria for Inoculation
3、Filter Paper Disks Containing of an Antimicrobic
4、Zone Diameter of Inhibition

24. Procedures
–Prepare a pure culture (18-24 hrs) of the sample on a non-selective medium
–Adjust turbidity until it is equivalent to the 0.5 McFarland Turbidity Standard
0.5 McFarland Standard
Sample

25. –Within 15 minutes of adjusting the turbidity
•dip a sterile cotton swab into the sample
•streak a lawn of bacteria on Mueller-Hinton agar
Leave the lid agar for 3-5 minutes (no more than 15 minutes) to allow plate to dry
Procedures

26. Procedures
–Apply antibiotic impregnated disks on the bacterial lawn
•Important: where the disk drops is where it stays
–Incubate for 16-18 hours at 33 2oC unless otherwise instructed

27. Results
–Antibiotics diffuse out onto the agar
–Concentration of antibiotics decrease as they diffuse further away from the disks
–After incubation, observe for a clearing on the bacterial lawn (zone of inhibition)
Bacterial growth
Zone of inhibition
incubation

28. Results
–Measure the diameters of the zone of inhibition
–Interpret the results as “resistant” or “susceptible” according to the guideline provided by the NCCLS
•Interpretation of the zone of inhibition is different for each bacteria-antibiotic combination

29. Disk Diffusion Test
•Qualitative results
–Susceptible
–Intermediate – may respond if infection is at body site where drug concentrates (e.g. urine) or if higher than normal dose can be safely given
–Resistant

30. Disk Diffusion
Test

31. Select colonies
Prepare inoculum
suspension

32. Mix well
Standardize inoculum
suspension

33. Swab plate
Remove sample

34. Add disks
Incubate overnight

35. Measure Zones

36. Modify methods for fastidious bacteria

37. Zone Interpretive Criteria (mm)
Drug
Disk content (ug)
Res
Int
Susc
cefazolin
30
 14
15-17
 18
gentamicin
10
 12
13-14
 15

38. Advantages and Disadvantages of Tests
Advantages :
1.easy to substitute one disk for another
2. dependent on a commercial provider for the drug profiles available
3. easier to spot contamination and low-level resistance
Disadvantages:
1. use only with rapidly growing organism
2. MBC can not be done using agar diffusion techniques

44. MIC on a strip
abbiodisk.com

45. E Test
 A new test method has been developed recently called the E Test, which is a modification of the disk diffusion test but provides an MIC result.
The MIC is read at the point where the zone intersects the MIC scale on the strip. Studies show this method to give greater than 95% agreement with the standardized broth microdilution method

46. Clinical Conditions when MICs are Useful
•Endocarditis
•Meningitis
•Septicemia
•Osteomyelitis
•Immunosuppressed patients (HIV, cancer, etc.)
•Prosthetic devices
•Patients not responding despite “S”

47. MIC
•Minimal inhibitory concentration
•The lowest concentration of antimicrobial agent that inhibits the growth of a bacterium
•Interpret:
–Susceptible
–Intermediate
–Resistant

48. E-test®
•Same principle as the Kirby-Bauer Test
•Uses a plastic strip with a predefined gradient of antibiotic concentration
•Results are read directly on the strip where the zone of inhibition intersects with the strip
E
Zone of inhibition
Bacterial growth

49. Results
•Interpret results as “resistant” or “susceptible” according to the guidelines provided in the package insert
•For ambiguous results, refer to the provided reading guide for :
–Organism related effects
–Drug related effects
–Resistance mechanism related effects
–Technical and handling effects

51. S. pneumoniae Penicillin MIC = 3 g/ml

52. MIC Interpretive Criteria (g/ml)
Drug
Susc
Int
Res
cefazolin
 8
16
 32
gentamicin
 4
8
 16

53. MICs Recommended (NCCLS M100-S14)
•Viridans Streptococcus – penicillin
•S. pneumoniae – penicillin, cefotaxime/ceftriaxone (sterile sites)
•Enterococcus – vancomycin “Int” results
•Staphylococcus – vancomycin zone 14 mm