Antimicrobial Chemotherapy and

Antibiotic Sensitivity TestingDr.T.V.Rao MD

11/1/2014 Dr.T.V.Rao MD 1

Uses of Antimicrobial Agents

• Antimicrobial agents are widely employed

to cure bacterial diseases

• Definition of Antibiotic – Antibiotics are

substances that are derived from a various

species of microorganisms and are

capable of inhibiting the growth of other

microorganism even in small

concentrations.

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Beginning of Antibiotics with

Discovery of Pencillin

• The discovery of penicillin

has been attributed to

Scottish scientist

Alexander Fleming in

1928 and the

development of penicillin

for use as a medicine is

attributed to the

Australian Nobel

Laureate Howard Walter

Florey.

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Discovery of Pencillin

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Discovery of Pencillin

Awarded Nobel Prize

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Chemotherapeutic Agents

• Antimicrobial agents – that are

produced synthetically but

have action similar to that of

antibiotics and are defined as

chemotherapeutic agents

• Eg Sulphonamides,

Quinolones.11/1/2014 Dr.T.V.Rao MD 6

Definition

• Bacteriostatic - Antimicrobial agents

that reversibly inhibit growth of

bacteria are called as bacteriostic (

Tetracyclnes, Chloramphenicol )

• Bactericical – Those with an

irreversible lethal action on bacteria

are known as bactericidal ( Pencillin,

Isoniazid )11/1/2014 Dr.T.V.Rao MD 7

How Drugs Act

• Drugs differ on their

capabilities to act at different

sites on bacteria.

• Some drugs have more than

one site of action

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Major mechanisms of

antimicrobial drugs

• 1 Inhibition of cell wall synthesis

• 2 Inhibition of cell membrane function

• 3 Inhibition of protein synthesis ( inhibition

of translation and transcription of genetic

material)

• 4 Inhibition of nucleic acid synthesis.

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Pencillin

• Pencillin and cephalosporins act inhibiting

Trans peptidases, the enzyme catalyses the final

linking step in synthesis of peptidoglycan.

• Due to this reason Pencillin in bactericidal for

grwoing bacteria since new peptidoglycan is

synthesized at that stage only.

• In nongrwoing cells pencillin is inactive

• An intact beta – lactum is essential for

antibacterial activity of pencillins

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Classification of Pencillins

• Natural

Benzyl pencillin

Phenoxymethyl pencillin Pencillin v

Semi synthetic and pencillase resistant

1 Methicillin

2 Nafcillin

3 Cloxacillin

4 Oxacillin

5 Floxacillin

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Extended spectrum pencillins

Aminopencillins - Ampicillin, Amoxycillin

Carboxypencillins – Carbencillin, Ticarcillin

Ureidopencillin - Piperacillin

Resistance to pencillin is due to pencillinase commonly called as ßlactamase

The enzyme opens Betalactam ring hydrolytically and thus converts the antibiotic to inactive pencillonic acid.

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Inhibitors to Beta lactamase

• Clavulinic acid which is a product of

Strept.clavuligerus

• Acts against the Staphylococcal beta

ßlactamase.

• And plasmid mediated Betalactamase of

Gram negative bacteria.

• Salbactum – this is a semisyntetic sulfone

derivative with weak antibacterial activity

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• Tazobactam – A penicillonic acid sulfone

derivative acts well when combined with

piperacillin.

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Cephalosporins• Like pencillin acts similar

• Products of the molds of genus

Cephalosporium except cefoxilin

• Divided into 4 generation of

cephalosporins depending on the

spectrum of activity.

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Different Generations of

Cephalosporins

• Cephalosporins are grouped into

"generations" based on their

spectrum of antimicrobial activity. The

first cephalosporins were designated

first generation while later, more

extended spectrum cephalosporins

were classified as second generation

cephalosporins.

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Major generations of Cephalosporins

• Cephalosporins are divided into 3 generations:

• 1st generation: Cephelexin, cefadroxil,

cephradine

• 2nd generation: Cefuroxime, cefaclor

• 3rd generation: cefotaxime, Ceftazidime,

cefixime - these give the best CNS penetration

• 4th generation Cephalosporins are already

available

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Basis of generations in

Cephalosporins

• Cephalosporins are grouped into

"generations" based on their spectrum of

antimicrobial activity. The first

cephalosporins were designated first

generation while later, more extended

spectrum cephalosporins were classified

as second generation cephalosporins.

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Advantages with Newer

generations

• Each newer generation of cephalosporins

has significantly greater gram-negative

antimicrobial properties than the preceding

generation, in most cases with decreased

activity against gram-positive organisms.

Fourth generation cephalosporins,

however, have true broad spectrum

activity

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Other Beta-lactams include

• Other beta-lactams include:

• Aztreonam: a monocytic beta-lactam,

with an antibacterial spectrum which

is active only against Gram negative

aerobes, including Pseudomonas

aeruginosa, Neisseria meningitidis

and N. gonorrhoea.

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Other drugs

• Imipenem: a carbapenem with a

broader spectrum of activity

against Gram positive and

negative aerobes and anaerobes.

Needs to be given with cilastatin

to prevent inactivation by the

kidney.

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Quinolones

• Quinolones are the first wholly synthetic

antimicrobials. The commonly used

Quinolones.

• Act on the DNA gyrase which prevents

DNA polymerase from proceeding at the

replication fork and consequently stopping

synthesis.

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Aminoglycosides

• Aminoglycosides are group of antibiotics in

which amino sugars liked by glycoside bonds

• Eg Streptomycin,

• Act at the level of Ribosome's and inhibits

protein synthesis

• Other Aminoglycosides –

Gentamycin,

neomycins,paromomycins,tobramycins

Kanamycins and spectinomycins

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Tetracycline's

• Broad spectrum antibiotic produced by

Streptomyces species

• 1. Oxytetracycle, chlortetracycle and

tetracycline

• Tetracyclnes are bacteriostatic drugs inhibits

rapidly multiplying organisms

• Resistance develops slowly and attributed to

alterations in cell membrane permeability to

enzymatic inactivation of the drug

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Choramphenicol

• Choramphenicol is

bacteriostatic drug

• Can produce bone marrow

depression

• Chloramphenicol interferes

with protein synthesis.11/1/2014 Dr.T.V.Rao MD 25

Macrolides,Azalides,Ketolides

• Contain macro cyclic lactone ring

Erythromycin. Is popularly used drug

• Other drugs

Roxithromycin,Azithromycin

• Inhibits the protein synthesis.

• Used as alternative to pencillin allergy

patients.

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Other Antimicrobial agents

• Lincomycins

Clindamycin resembles Macrolides in

biting site and antimicrobial activity.

Streptogramins

Quinpristin / dalfopristin

useful in gram postive bacteria

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Antibiotics in Anaerobes

• Major anaerobes – Anaerobic cocci, clostridia and Bacteroides are susceptible to Benzyl pencillin

• Bact.fragilis as well as many other anaerobes are treatable with Erythromycin,Lincomycin, tetracycline and Chloramphenicol

• Clindamycin is effective against many strains of Bacteroides

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Metronidazole in Anaerobic

Infections• Since the discovery of Metronidazole in

1973 since then it was identified as

leading agent anaerobes.

• But also useful in treating parasitic

infections

Trichomonas, Amoebiasis and other

protozoan infections.

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Antiviral drugs

• Acyclovir

• Ribavirin

• Azidothymidine

• Amantidine

• Idoxuridine

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Antifungal Agents

• Grisofulvin

• Imidazole

• Polyenes Nystatin, Amphotericin B

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Antibiotic resistance

• Antibiotic resistance is the ability of a micro

organism to withstand the effects of antibiotics. It

is a specific type of drug resistance. Antibiotic

resistance evolves naturally via natural selection

acting upon random mutation, but it can also be

engineered by applying an evolutionary stress

on a population. Once such a gene is generated,

bacteria can then transfer the genetic

information in a horizontal fashion (between

individuals) by plasmid exchange.

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Detection of Antibiotic sensitivity and Resistance

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Routine Susceptibility Tests

• Disk diffusion

(Kirby Bauer)

• Broth micro-

dilution MIC

– NCCLS reference

method

• Etest

Dr.T.V.Rao MD 3411/1/2014

• Diffusion method

– Put a filter disc, or a porous cup/a bottomless

cylinder containing measured quantity of

drugs on the a solid medium that has been

seeded with test bacteria

• Dilution method

– vary amount of antimicrobial substances

incorporated into liquid or solid media

– followed by inoculation of test bacteriaDr.T.V.Rao MD 35

Antimicrobial Susceptibility

Testing

11/1/2014

Susceptibility Testing

Methods

Innoculate

MH plate

Place disks

on agar plate

Incubate plate

18-24 hr, 35 C

Measure and

record zone of

inhibition around

each disk11/1/2014 Dr.T.V.Rao MD 36

Antimicrobial susceptibility testsMinimum inhibitory concentration [MIC]

– The smallest concentration of antibiotic that

inhibits the growth of organism

Liquid media (dilution) allows MIC estimation

Solid media (diffusion)

– Disk diffusion (Kirby-Bauer)

– E-tests

– Allows MIC estimation

Beta lactamase production: quick screening

method11/1/2014 Dr.T.V.Rao MD 37

Disk Diffusion

Susceptibility Testing

Improper agar & disk placement Mueller Hinton agar &

good disk placement

Use Mueller Hinton agar

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Antimicrobial susceptibility

testing using micro-broth

dilutions

•

•

•

•

•

•

•

• •

•

•

•

•

96 well microtiter plate

ug/ml

64 32 16 8 4 2

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Dilution in liquid broth

• Tubes containing increasing antibiotic

concentrations

• Incubation during 18 hr at 37°C

• Tedious

0 (Control) 0,25 0,50 1 2 4 8 mg/l

MICBacterial growth Inhibition

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Kirby-Bauer disc testingAntibiotic-impregnated discs placed on an agar plate at the

interface between test organism and susceptible control organism

Resulting zones of inhibition compared, use of controls

Susceptibility is inferred (standard tables)

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Antimicrobial Gradient

Testing

E-test®

Read plates

after

recommended

Incubation

Read MIC

where elipse

intersects

scale

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Dr.T.V.Rao MD 43

E test – MIC Reports are helpful in

Critical management decisions

• Quantitative MIC

data is a

prerequisite for

the management

of critical

infections,

including sepsis,

especially among

critical care

patients. Etest is

11/1/2014

E-testPlastic strips with a predefined

gradient of

– One antibiotic

– One antifungal

Only one manufacturer

One strip per antibiotic

Wide range of antibiotics

Easy to use

Storage at -20°C

Short shelf life, expensive

Reading E-tests

Susceptible < 1

Resistant > 4 ug/ml

Ciprofloxacin for

Yersinia pestis

Intermediate 1-4 ug/ml

Upper reading

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Are we overusing Antibiotics

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Choose the Appropriate

Antibiotic

Think before

prescribing

Are we using

Right drug

for the Right

bug ?

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•Program created by Dr.T.V.Rao MD for Medical and Paramedical Students in

the Developing world

• Email.

• doctortvrao@gmail.com

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