Antibiotic susceptibility testing بكتريا عملي
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The document discusses antimicrobial susceptibility testing. It begins by defining antibiotics and classifying different types of antibiotics based on their mechanism of action and targets, such as cell wall synthesis inhibitors and protein synthesis inhibitors. It then describes two common methods for antimicrobial susceptibility testing - the disk diffusion method and Etest. The disk diffusion method involves measuring the zone of inhibition around disks containing different antibiotics. The Etest provides minimum inhibitory concentration (MIC) values by using plastic strips with gradients of antibiotic concentrations. Interpretive criteria are used to determine if a bacteria is susceptible, intermediate, or resistant to a given antibiotic.
Antibiotic susceptibility testing 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
- 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