Antibiotic susceptibility testing بكتريا عملي
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Transcript of Antibiotic susceptibility testing بكتريا عملي
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Antimicrobial Susceptibility Testing
By
Dr. Nabil El Aila
Assistant Professor of Molecular Microbiology
Medical Technology Department
Al -Aqsa University
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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
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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
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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
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Classification
Beta-lactams Poly-peptides
Cell wall synthesis inhibitors
Penicillins
Cephalosporins
Monobactam (Aztreonam)
Carabpenems
(Meropenem/Imipenem)
Bacitracin
Vancomycin
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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
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β-Lactams
Dr. Nabil El Aila General Microbiology
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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
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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
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Classification
Aminoglycosides
Protein synthesis inhibitors
Tetracyclines
Macrolides
Clindamycin
Chloramphenicol
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Classification
Aminoglycosides
Protein synthesis inhibitors
• Gentamicin
• Tobramycin
• Streptomycin
• Neomycin
• Kanamycin
• Amikacin
Tetracyclines Macrolides
• Tetracycline
• Doxytetracycline
• Minocycline
• Doxycycline
• Erythromycin
• Azithromycin
• Clarithromycin
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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
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Classification
Sulfonamide
Folate antagonist
• Sulfadiazine
• Sulfadimidine
• Sulfamethoxazole
Quinolones Trimethoprim
• Nalidixic acid
• Ciprofloxacin
• Levofloxacin
• Ofloxacin
• Norfloxacin
• Travofloxacin
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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
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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
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Dr. Nabil El Aila General Microbiology
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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
!!!
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Routine Susceptibility Tests
• Disk diffusion (Kirby Bauer)
• Etest
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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.
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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.
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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
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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
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– 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
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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
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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
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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
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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
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Disk Diffusion
Test
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Select colonies
Prepare inoculum suspension
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Mix well
Standardize inoculum suspension
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Swab plate
Remove sample
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Add disks
Incubate overnight
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Measure Zones
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Modify methods for fastidious bacteria
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Zone Interpretive Criteria (mm)
Drug
Disk
content
(ug)
Res
Int
Susc
cefazolin 30 14 15-17 18
gentamicin 10 12 13-14 15
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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
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MIC on a strip abbiodisk.com
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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
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Clinical Conditions when MICs are Useful
• Endocarditis
• Meningitis
• Septicemia
• Osteomyelitis
• Immunosuppressed patients (HIV, cancer, etc.)
• Prosthetic devices
• Patients not responding despite “S”
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MIC
• Minimal inhibitory concentration
• The lowest concentration of antimicrobial agent that inhibits the growth of a bacterium
• Interpret:
– Susceptible
– Intermediate
– Resistant
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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
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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
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S. pneumoniae Penicillin MIC = 3 g/ml
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MIC Interpretive Criteria (g/ml)
Drug
Susc
Int
Res
cefazolin 8 16 32
gentamicin 4 8 16
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MICs Recommended (NCCLS M100-S14)
• Viridans Streptococcus – penicillin
• S. pneumoniae – penicillin, cefotaxime/ceftriaxone (sterile sites)
• Enterococcus – vancomycin “Int” results
• Staphylococcus – vancomycin zone 14 mm