Methods of drug testing, Drug

resistance & Vaccine introduction

Dr Shristi Ram
2-11-2021

 Methods of drug testing

Disk di usion methods
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 Minimum inhibitory concentration

• Determination of e ectiveness, Lowest conc of

drug which prevents the growth of particular
pathogen
• Minimum Bactericidal Concentration
(MBC)Minimum bactericidal concentration (MBC)
is de ined as the lowest concentration of antibiotic
that kills 99.9% of the inoculum.
• Also termed as minimum lethal concentration
(MLC), MBC of an antibacterial agent is determined
by subculturing last clear MIC tube onto growth
medium and examining for bacterial growth.
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 Kirby bauer method

• Developed in the early 1960s by W Kirby, AW Bauer and their
colleagues
• Most common antimicrobial susceptibility test. 
• This method relies on the inhibition of bacterial growth measured
under standard conditions.
• For this test, a culture medium, speci ically the Mueller-Hinton agar,
is uniformly and aseptically inoculated with the test organism and
then ilter paper discs, which are impregnated with a speci ic
concentration of a particular antibiotic, are placed on the medium.
•  The organism will grow on the agar plate while the antibiotic
“works” to inhibit the growth. 
• If the organism is susceptible to a speci ic antibiotic, there will be
no growth around the disc containing the antibiotic.
• Thus, a “zone of inhibition” can be observed and measured to
determine the susceptibility to an antibiotic for that particular
organism.
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 E-test method

• A plastic strip with a prede ined

gradient of one antibiotic is
applied onto an inoculated agar
plate.   
• After 18-24 hours incubation, a
drop-shaped inhibition zone
intersects the graded test strip at
the inhibitory concentration of the
antibiotic.

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 Bacterial Warfare
Bacteria must compete with each other to
survive.
One bacterial defense is to secrete toxins!

Antibiotics are these same toxins produced in

mass quantities to kill bacteria that harm
mammalian cells.
 The First Antibiotic

Penicillium

The first antibiotic was discovered by Alexander

Fleming in 1928 when he noticed that the fungus
penicillium killed disease causing bacteria.
 Antibiotic Resistance Facts

Antibiotic resistance is the process by which

bacteria become resistant to antibiotics.

Humans can have allergic reactions to

antibiotics, but we do not become resistant
to antibiotics.
Antibiotic Resistance Crisis
 Drug resistance
• Reduced permeability to antibiotic: Many G- bacteria are resistant to penicillin G because
drug fail to penetrate the cell membrane. Mycobacteria resist many drugs because of its high
mycolic acid content in LPS.
• E lux (pumping out of the cell): E. coli, Pseudomonas aeruginosa, Mycobacterium smegmatis,
M. tuberculosis have e lux pumps.
• Drug inactivation through chemical modi ication: Eg. hydrolysis of beta-lactam ring by
penicillinase. Chloramphenicol acetyltransferase-inactivates the drug by acetylation using
acetyl co-A as the donor.
• Target modi ication: Target enzyme or organ is modi ied, change in 23S rRNA decreases the
a inity of erythromycin and chloramphenicol to bind. Mycobacterium becomes resistant to
rifamcin by altering the beta subunit of its RNA polymerase
• Development of a resistance biochemical pathway: the microbe may use alternate pathway.
Bacteria show resistance to sulphonamide by obtaining folic acid from surrounding rather then
synthesising it.
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Mechanism of DR
 Origin of drug resistance

• Chromosome- mediated DR: Spontaneous mutation, usually change in the

drug receptors so antibiotics cannot bind.
• Transposons: carry antibiotic resistance gene (Tn5- kanamycin,
bleomycin, streptomycin), Tn21 (tetracycline), Tn 551 (erythromycin)
• Plasmid mediated DR: R plasmids, irst found in enterobacteria. R genes
encode, enzymes which degrade antibiotics or actively pump it out of the
cell.
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 Spread of DR
• Drug misuse: prescribed without culturing or identifying, self- precription,
not completing the antibiotic dosage.
• Extensive drug treatment: help in spreading of DR strains. May lead to
superinfection, pseudomembranous enterocolitis caused by Clostridium
di icile.
• Movement of resistance genes: Movement of transposons elements,
resistance genes, integron,
• Use of animal feed: farmyard antibiotics, Synercid, has led to increase in
vancomycin and synercid resistance in enterococci.
• Use of triclosan: antibacterial agents in soap, deo, mouth washes, baby
toys.
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 How to encounter DR
• Strategic use of drugs: high conc to eradicate the pathogen; mixture of
drugs simultaneously; chemotherapeutic drugs (broad spectrum) to be
used only when necessary
• Search for new antibiotics: “enhancers” , cationic peptide that disrupt
bacterial cell membrane by displacing Mg++.
• Identifying new targets for drugs: targeting new enzymes from di erent
metabolic pathways.
• Phage therapy: bacteriophage used to treat bacterial disease. In Russia,
Bandages are saturated with phage solution. Phage mixture are
administered orally. Staphylococcus infection are being treated using this
technique.

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 Vaccine

• Edward Jenner

• In May 1796, Edward Jenner found a young dairymaid, Sarah Nelms, who
had fresh cowpox lesions on her hands and arms. Using matter from
Nelms' lesions, he inoculated an 8-year-old boy, James Phipps.
Subsequently, the boy developed mild fever and discomfort in the axillae.
Nine days after the procedure he felt cold and had lost his appetite, but on
the next day he was much better.
• In July 1796, Jenner inoculated the boy again, this time with matter from a
fresh smallpox lesion. No disease developed, and Jenner concluded that
protection was complete.

Louis Pasteur- The first live attenuated

vaccines
• Pasteur was studying chicken cholera (Pasteurella multocida),
a diarrhoeal disease that was destroying the breeding chicken
population.
• Influenced by Edward Jenner, Pasteur reasoned that if a
vaccine could be found for smallpox, vaccines could be found
for all diseases.
• Pasteur began to study chicken cholera in 1877 and by the
following year had succeeded in culturing the causative
organism, Pasteurella multocida.
• In 1879, Pasteur discovered by chance that cultures of this
bacterium gradually lost their virulence over time.
• Before leaving to go on a holiday, Pasteur had instructed an
assistant to inject the latest batch of chickens with fresh
cultures of P. multocida.
• The assistant forgot to do this, however, and then himself went
on holiday.
• On his return, Pasteur’s assistant inoculated the chickens with
the cultures, which by this time had been left in the laboratory
for a month, stoppered only with a cotton-wool plug.
• The inoculated chickens developed mild symptoms but
recovered fully.

 An ideal vaccine
• Should be safe (not tumourogenic/ toxic/pathogenic)
• Should have very low levels of side e ects
• Should be safe for individuals with impaired immune system
• Should not spread or be contagious
• Should induce long lasting humeral and cellular immunities
• Vaccination techniques should be simple
• Should be cheap and a ordable
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 Two Types of Immunization

• Passive Immunization
• Methods of acquisition include natural maternal antibodies, antitoxins, and immune
globulins
• Protection transferred from another person or animal

• Active Immunization
• Methods of acquisition include natural infection, vaccines (many types), and toxoids
• Relatively permanent

 Passive Immunization

• Can occur naturally via transfer of maternal antibodies across placenta to fetus

• Injection with preformed antibodies

• Human or animal antibodies can be used
• Injection of animal Ab’s prevalent before vaccines

• E ects are only temporary

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 Conditions Warranting Passive Immunization

1. De iciency in synthesis of Ab as a result of congenital or acquired B-cell defects

2. Susceptible person is exposed to a disease that will cause immediate complications

(time is the biggest issue)

3. Disease is already present

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The Immune System and Passive Immunization

• The transfer of antibodies will not trigger the immune system

• There is NO presence of memory cells

• Risks are included

• Recognition of the immunoglobulin epitope by self immunoglobluin paratopes
• Some individuals produce IgE molecules speci ic for passive antibody, leading to mast cell degranulation
• Some individuals produce IgG or IgM molecules speci ic for passive antibody, leading to hypersensitive
reactions

 Active Immunization

• Natural Infection with microorganism or arti icial

acquisition (vaccine)

• Both stimulate the proliferation of T and B cells,

resulting in the formation of e ector and memory
cells

• The formation of memory cells is the basis for the

relatively permanent e ects of vaccinations

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