antibiotic susceptibility testing بكتريا عملي

Antimicrobial Susceptibility Testing

By

Dr. Nabil El Aila

Assistant Professor of Molecular Microbiology

Medical Technology Department

Al -Aqsa University

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

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

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

Classification

Beta-lactams Poly-peptides

Cell wall synthesis inhibitors

Penicillins

Cephalosporins

Monobactam (Aztreonam)

Carabpenems

(Meropenem/Imipenem)

Bacitracin

Vancomycin

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

β-Lactams

Dr. Nabil El Aila General Microbiology

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

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

Classification

Aminoglycosides

Protein synthesis inhibitors

Tetracyclines

Macrolides

Clindamycin

Chloramphenicol

Classification

Aminoglycosides

Protein synthesis inhibitors

• Gentamicin

• Tobramycin

• Streptomycin

• Neomycin

• Kanamycin

• Amikacin

Tetracyclines Macrolides

• Tetracycline

• Doxytetracycline

• Minocycline

• Doxycycline

• Erythromycin

• Azithromycin

• Clarithromycin

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

Classification

Sulfonamide

Folate antagonist

• Sulfadiazine

• Sulfadimidine

• Sulfamethoxazole

Quinolones Trimethoprim

• Nalidixic acid

• Ciprofloxacin

• Levofloxacin

• Ofloxacin

• Norfloxacin

• Travofloxacin

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

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

Dr. Nabil El Aila General Microbiology

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

!!!

Routine Susceptibility Tests

• Disk diffusion (Kirby Bauer)

• Etest

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.

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.

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

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

– 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

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

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

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

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

Disk Diffusion

Test

Select colonies

Prepare inoculum suspension

Mix well

Standardize inoculum suspension

Swab plate

Remove sample

Add disks

Incubate overnight

Measure Zones

Modify methods for fastidious bacteria

Zone Interpretive Criteria (mm)

Drug

Disk

content

(ug)

Res

Int

Susc

cefazolin 30 14 15-17 18

gentamicin 10 12 13-14 15

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

MIC on a strip abbiodisk.com

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

Clinical Conditions when MICs are Useful

• Endocarditis

• Meningitis

• Septicemia

• Osteomyelitis

• Immunosuppressed patients (HIV, cancer, etc.)

• Prosthetic devices

• Patients not responding despite “S”

MIC

• Minimal inhibitory concentration

• The lowest concentration of antimicrobial agent that inhibits the growth of a bacterium

• Interpret:

– Susceptible

– Intermediate

– Resistant

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

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

S. pneumoniae Penicillin MIC = 3 g/ml

MIC Interpretive Criteria (g/ml)

Drug

Susc

Int

Res

cefazolin 8 16 32

gentamicin 4 8 16

MICs Recommended (NCCLS M100-S14)

• Viridans Streptococcus – penicillin

• S. pneumoniae – penicillin, cefotaxime/ceftriaxone (sterile sites)

• Enterococcus – vancomycin “Int” results

• Staphylococcus – vancomycin zone 14 mm