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This is a series of lectures on microbiology useful for undergraduate medical and paramedical students
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ANTI MICROBIAL CHEMOTHERAPY
Dr. Ashish Jawarkar
M.D.
Consultant Pathologist
Parul Sevashram Hospital
Modern era in Antibiotics begins with Fleming.
Definition - Antibiotic
Anti – against Bio – life Originally extracted from compounds produced by
living organisms – aminoglycosides Semi synthetic – cephalosporins, carbapenems Synthetic – sulphonamides, quinolones,
oxazolidinediones
History of Antibiotic Discovery
Mechanisms of antibacterial action
1. Inhibition of cell wall synthesis Penicillin, vancomycin, bacitracin
2. Inhibition of protein synthesis Aminoglycosides, tetracyclines, chloramphenicol, macrolides
3. Alteration of cell membranes Polymyxin B, Bacitracin
4. Inhibition of nucleic acid synthesis DNA – quinolones, Metronidazole RNA – Rifampicin, Bacitracin
5. Antimetabolite activity Sulfonamides
Antimicrobial Resistance
How bacteria survive doses of antibiotics that would otherwise have been lethal
Problem – treated with a higher dose, or newer antibiotic
Side effects, Newer may not be available
Types of resistance
Intrinsic Bacteria may not make the protein that antibiotic
targets
Extrinsic Due to acquired mutation in DNA or RNA
Causes of Resistance
Overuse/misuse of antibiotics Example is sore throat – Viral , still antibiotics are
prescribed
Failure to take entire course Failure to take at correct intervals – two times a day
instead of three times
Antibiotic Susceptibility Testing
Pathogenic bacteria show large variations in susceptibility to antibiotics
Particularly marked in case of Staphylococcus and gram negative bacteria
Types
Diffusion tests Dilution tests
Uses of Antibiotic Sensitivity Testing
Antibiotic sensitivity test: A laboratory test which determines how effective antibiotic therapy is against a bacterial infections.
Antibiotic sensitivity testing will control the use of Antibiotics in clinical practice
Testing will assist the clinicians in the choice of drugs for the treatment of infections.
Components of Antibiotic Sensitivity Testing
1.The identification of relevant pathogens in exudates and body fluids collected from patients
2. Sensitivity tests done to determine the degree of sensitivity or resistance of pathogens isolated from patient to an appropriate range of antimicrobial drugs
Antibiotic Sensitivity Testing Is Essential of selection of
Antibiotics
Isolation and Identification of Bacteria precedes the selection of Antibiotic
Testing Methods
Uses of Antibiotic Sensitivity Testing
Helps to guide the Physician in choosing Antibiotics
The accumulated results on different pathogens their sensitivity will guide the physician in choosing empirical treatment in serious patients before the individual’s laboratory results are analyzed in the Microbiology laboratory.
Reveals the changing trends in the local isolates.
Why Need continues for testing for Antibiotic Sensitivity
Bacteria have the ability to develop resistance following repeated or subclinical (insufficient) doses, so more advanced antibiotics and synthetic antimicrobials are continually required to overcome them.
Testing for Antibiotic sensitivity
The method includes several steps including obtaining a bacterial sample;
identifying the type of bacteria in the bacterial sample; selecting a set of antibiotics based on the identity of the bacteria in the bacterial sample;
obtaining a control sample from the bacterial sample;
What is Resistance in Antibiotic Sensitivity Testing
Resistance implies that the infection is not treatable with the tested Antibiotic because its MIC exceeds achievable safe tissue or urine levels.
Kirby-Bauer methods A commonly used method in basic
laboratories Kirby-Bauer antibiotic testing (KB testing or
disk diffusion antibiotic sensitivity testing) is a test which uses antibiotic-impregnated wafers to test whether particular bacteria are susceptible to specific antibiotics
How to perform Kirby- Bauer testing
The basics are easy: The bacterium is swabbed on the agar and the antibiotic discs are placed on top. The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is away from the disc. If the organism is killed or inhibited by the concentration of the antibiotic, there will be NO growth in the immediate area around the disc: This is called the zone of inhibition .
Steps in Antibiotic sensitivity testing
Bacteria are inoculated as lawn culture
Method of inoculation- Good results are obtained by placing a standard loopful of inoculum suspension on the plate and then spreading it with a dry sterile swab.
The area of Inhibition is measured with a Scale
Record the results for everyone on your table in the table below.
The disk diffusion methods are commonly used for routine testing
Read the plates in transmitted light
The zone of inhibition guides the right choice of
Antibiotic
The strips with multiple Antibiotics can be tested in one go
Other methods of Antibiotic susceptibility testing
Other methods to test antimicrobial susceptibility include the Stokes method, E-test (also based on antibiotic diffusion). Agar and Broth dilution methods for Minimum Inhibitory Concentration determination.
Testing Minimum Inhibitory Concentration
In alternative measure of susceptibility is to determine the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of a drug. A series of broths are mixed with serially diluted antibiotic solutions and a standard inoculum is applied. After incubation, the MIC is the first broth in which growth of the organism has been
What is Minimum Inhibitory
concentration Minimum inhibitory concentration (MIC), in microbiology, is the lowest concentration of an antimicrobial that will inhibit the visible growth of a micro organism after overnight incubation. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of micro organisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents.[
The Antibiotics are diluted to various dilution to test the minimum
inhibitory concentration
What is E Test
Etest is an antimicrobial gradient technique in which 15 reference MIC dilutions of an antibiotic have been repackaged with innovative dry chemistry technology onto a plastic strip. The predefined gradient provides precise and accurate assessment of antimicrobial activity against both fastidious and non-fastidious microorganisms.
Dr.T.V.Rao MD
The strips are impregnated with various
concentration of Antibiotics
E = testing on various isolates
Limitation of Disk Diffusion Methods
Disk diffusion methods are not suitable for slow growing bacterial pathogens
The great limitation being for testing on Mycobacterium tuberculosis which needs varied, technically demanding methods
Antibiotic Sensitivity testing can be done with automation
There is a growing need for Automation in Antibiotic sensitivity
testing
Limitation of Antibiotic Sensitivity usage
Both Microbiologists and Clinician should however bear in mind that the response therapy in vivo may
not always reflect the results of testing the sensitivity of patient's pathogen in vitro.
Thank You