Antibiotics

1. Sreenu Thalla
Associate Professor
Department of Pharmacology
Antibiotics

2. Antibiotics are the drugs that are commonly used to fight against infections caused
by bacteria.
•3 - 40% of patients admitted to hospital acquire an infection during their stay, and that the
risk for hospital-acquired infection, or nosocomial (hospital acquired) infection, has risen
steadily in recent decades.
•The frequency depends mostly on the type of conducted operation being greater for “dirty”
operations (10-40%), and smaller for “pure” operations (3-7%).
E.g. such serious infectious complication as postoperative meningitis is often the result of
nosocomial infection.
• According to the Center for Disease Control and Prevention (CDC) statistics, more than 70%
of the bacteria that cause hospital-acquired infections are resistant to at least one of the
antibiotics most commonly used to treat infections.
Antibiotics

3. • Analysis of the microbiological data included in antibiograms collected in different
institutions over different periods of time is considered as one of the most important
activities to restrain the spreading of antibiotic resistance and to avoid the negative
consequences of this phenomenon
How antibiotics work ?
• Inhibition of nucleic acid synthesis
– Rifampicin; Chloroquine
• Inhibition of protein synthesis
– Tetracyclines; Chloramphenicol
• Action on cell membrane
– Polyenes; Polymyxin
• Interference with enzyme system
– Sulphamethoxazole
• Action on cell wall
– Penicillin; Vancomycin

4. Antibiotic sensitivity of different bacteria
• Comparing the antibiotic sensitivity of different bacteria

5. The emergence of antibiotic resistance
 Effects of different antibiotics on growth of a Bacillus strain.
 The right-hand image shows a close-up of the novobiocin disk (marked by an arrow
on the whole plate).
 In this case some individual mutant cells in the bacterial population were resistant to
the antibiotic and have given rise to small colonies in the zone of inhibition.

6. How Antibiotic Resistance Happens

7. How Antibiotic Resistance Happens
• In spontaneous DNA mutation, bacterial DNA may
mutate spontaneously. Drug-resistant tuberculosis
arises this way.
• In a form of microbial sex called transformation, one
bacterium may take up DNA from another bacterium.
Pencillin-resistant gonorrhea results from
transformation.
• Resistance acquired from a small circle of DNA called
a plasmid, that can flit from one type of bacterium to
another.
– A single plasmid can provide a slew of different
resistances.
– In 1968, 12,500 people in Guatemala died in an
epidemic of Shigella diarrhoea. The microbe
harbored a plasmid carrying resistances to four
antibiotics!

8. How Antibiotic Resistance Happens
• Horizontal Gene Transfer (© Grace Yim and Fan Sozzi)

9. Mechanisms of Antibiotic Resistance
9

10. 10
Antibiotic Method of resistance
Chloramphenicol reduced uptake into cell
Tetracycline active efflux from the cell
β-lactams, Erythromycin, Lincomycin
eliminates or reduces binding of antibiotic to
target
β-lactams, Erythromycin hydrolysis
Aminoglycosides, Chloramphenicol,
Fosfomycin, Lincomycin
inactivation of antibiotic by enzymatic
modification
β-lactams, Fusidic Acid sequestering of the antibiotic by protein binding
Sulfonamides, Trimethoprim metabolic bypass of inhibited reaction
Sulfonamides, Trimethoprim overproduction of antibiotic target (titration)
Bleomycin
binding of specific immunity protein to
antibiotic

11. Problem Formulation
• More global, e.g. for pharmaceutical companies
– Maintain a pool of effective drugs on the market
• Research, develop and test new antimicrobials
• Widespread misuse of antibiotics
• More local, e.g. for a hospital
– Maintain a pool of effective drugs in the hospital
• Monitoring and researching … (concept drift, seasons)
– Predicts the sensitivity of certain antibiotic for a certain patient with a certain
disease
• Various intelligent techniques including KM, KDD and DM, ML, DSS etc.

12. 12
Patterns of antibiotic resistance.

13. Grouping of Pathogens
Pathogen group S 1 S 2 S 3 Total Total %
All 1967 217 2244 4430 100%
Gram + 1 089 43 1 002 2 134 48,2%
Gram - 880 174 1 242 2 296 51,8%
Staf _g+ 1 028 43 942 2 013 45,4%
Enterococ_g+ 61 0 60 121 2,7%
Enterobac_g- 237 60 486 783 17,7%
Nonferm_g- 643 114 756 1 513 34,2%

14. Grouping of Antibiotics
Ant_group S 1 S 2 S 3 total total%
Pen _ingib 144 29 148 321 7,2%
Monobact 28 7 56 91 2,1%
Glyco 179 0 2 181 4,1%
Amino 309 34 346 689 15,6%
Fhinolon 212 39 261 512 11,6%
Sulph 122 0 186 308 7,0%
Macrolydes 38 3 124 165 3,7%
Tetra 43 2 136 181 4,1%
Rif 132 6 26 164 3,7%
Nitrofuran 71 5 136 212 4,8%
Lactases 167 1 14 182 4,1%
Fuzid 89 2 20 111 2,5%
b -lactam 435 89 789 1 313 29,6%
Ceph 183 78 292 553 12,5%
c_penem 116 2 19 137 3,1%
Pen 136 9 478 623 14,1%

15. Microbiological assays
It is based on comparison of inhibition of microbial growth by measured
concentration antibiotic to be examined with that produced by known concentrations of
a standard preparation of the antibiotic having known activity
Methods
1. Cylinder plate (or) Cup-Plate method
2. Turbidometric (or) Tube assay method
 Cylinder plate (or) Cup-Plate method is based on DIFFUSION of antibiotic from vertical
cylinder into media containing organisms
 Turbidometric method is based on INHIBITION of microbial growth in an antibiotic
solution

16. Essentials
1. Apparatus
2. Standard preparation
3. Culture media
4. Buffer solution
5. Test organism
6. Preparation of standard and sample solution
7. Inoculam preparation
8. Measurements
9. Assay
10. Precision

17. 1. Apparatus
Cylinder method –  Petri dishes (20 × 100mm)
 Assay cylinder – outside diameter (8mm ± 0.1mm)
inside diameter (6mm ± 0.1mm)
length (10mm ± 0.1mm)
 Paper discs (or) sterile borer
Turbidometric method – Glass (16 × 125mm)
(or)
Plastic tubes (18 × 150mm)
2. Standard preparation
Sample of antibiotic potency determined with reference to the international
standard

18. Stock solution and test dilutions of standard preparations
Standard stock soltion Test dilution
Antibiotic Assay
method
Prior
drying
Initial
solvent
Final stock
solution
(mg/ units)
Use
before
(days)
Final
diluent
Median
dose
(µg/ml)
Incubation
temperature
Amikacin B NO Water 1mg 14 Water 10 32-35
Amphoterici
n-B
A YES DMF 1mg Same
day
B5 1.0 29-31
Bacitracin A YES 0.01M
HCl
100
units
Same
day
B1 1.0 32-35
Gentamycin A YES B2 1mg 30 B2 0.1 36-37.5
Rifampicin A NO Methanol 1mg 1 B1 5 29-31
Streptomycin A4 YES Water 1mg 30 Water 1 32-35
Tetracycline A3 NO 0.1M
HCl
1mg 1 Water 2.5 32-35

19. 3. Culture media
Dissolve ingredients in water to produce 1000ml and pH adjusted + sufficient
1M NaOH (or) 1M Hcl
Ingredient Medium type
A B C D E F G H I J
Peptone 6 6 5 6 6 6 9.4 - 10.0 -
Pancreatic digest 4 - - 4 - - - 17 - 15
Yeast extract 3 3 1.5 3 3 3 4.7 - - -
Beef extract 1.5 1.5 1.5 1.5 1.5 1.5 2.4 - 10 -
Dextrose 1 - 1 1 - - 10 2.5 - -
Soyabean digest - - - - - - - 3 - 5
Composition

20. Ingredient
Medium type
A B C D E F G H I J
Agar 15 15 - 15 15 15 23.5 12 17 15
Glycerin - - - - - - - - 10 -
Polysorbate 80 - - - - - - - 10 - -
Sodium chloride - - 3.5 - - - 10 5 3 5
Dipotassium
hydrogen phosphate
- - 3.68 - - - - 2.5 - -
Potassum disodium
hydrogen phosphate
- - 1.32 - - - - - - -
Final pH 6.5
–
6.6
6.5
–
6.6
6.95
–
7.05
7.8
–
8.0
7.8
–
8.0
5.8
–
6.0
6.0
–
6.2
7.1
–
7.3
6.9
–
7.1
7.2
–
7.4

21. 4. Buffer solution
The ingredients were dissolved in sufficient water to produce 1000ml and PH
adjusted
Buffer number Di-potassium
hydrogen phosphate
Potassium di-
hydrogen phosphate
PH
1 2 8 6 ± 0.1
2 16.73 0.523 8 ± 0.1
3 - 13.61 4.5 ± 0.1
4 20.0 80.00 6 ± 0.1
5 35.0 - 10.5 ± 0.1
6 13.6 4.0 7 ± 0.2

22. 5. Test organism
To be maintained in a culture on the slants of the medium and transferred to
fresh slants after incubation.
S.No Antibiotic Test organism
1 Amikacin Staphylococcus auries
2 Amphotericin – B Saccharomyces cerevisiae
3 Bacitracin Micrococcus luteus
4 Doxycycline Staphylococcus auries
5 Erythromycin Staphylococcus auries
6 Gentamycin Staphylococcus epidermi
7 Kanamycin sulphate Bacillus pumilus
8 Kanamycin – B Bacillus subtilics
9 Nystatin Saccharomyces cerevisiae
10 Rifampicin Bacillus subtilis
11 Streptomycin Bacillus subtilis and Klebsella pneumoniae
12 Tetracycline Bacillus cereus and Staphylococcus auries

23. 6. Preparation of standard and sample solution
 Dilution of standard preparation to
required concentration to prepare
a stock solution
 Dissolve a quantity of standard
preparation of given antibiotic,
weighed and dried.
 Assign an assumed potency / unit
weight or volume for substance being
examined and prepare stock solution
and test dilution

24. 7. Inoculam preparation
Test organism Incubation conditions Suggested
dilution
factor
Inoculam composition
Medium
method
Temp Time Medium Amou
nt/ml
Antibiotic
assayed
Staphylococcus
aureus
A/1 32-35 24hr 1 : 20 C 0.1 Amikacin,
Doxycycline,
Tetracycline
Kanamycin sulphate
Saccharomyces
cerevisiae
G/3 23-31 48hr As
determined
G 1.0 Amphotericin – B,
Nystatin
Micrococcus
luteus
A/1 32-35 24hr 1 : 35 A 0.3 Bacitracin
Micrococcus
luteus
A/1 32-35 24hr 1 : 40 D 1.5 Erythromycin
Staphylococcus
epidermidis
A/1 32-35 24hr 1 : 40 D 0.03 Gentamycin
Bacillus subtilis A ½ 32-35 5days - E,E,B As
read
Franmycitin – E
Kanamycin – E
Rifampicin
Klebsiella
pneumoniae
A/1 36-37 24hr 1 : 25 C 0.1 Streptomycin

25. For method – A
 The inoculam was prepared and mixed with 100ml of medium and the plates were
prepared using this inocula.
 Double layer plates may be prepared by pouring the seed layer inoculated with the desired
micro organism over a solidified un-inoculated base layer.
Eg : 21ml base layer
4ml seed layer
 Each cylinder was filled with a median concentration of antibiotic and incubate the plates.
 After incubation examine and measure the “ Zone of inhibition”
For method – B
 The procedure adopted is described for the specific antibiotic assay running only the high
and low concentration of the standard response curve.
 After incubation the absorbance of the apparatus tubes measured

26. 8. Measurement
Cylinder plate method - diameter or area of the circular inhibition zone is measured
Turbidimetric method – spectrophotometer is used to measure the transmittance
TEMPERATURE CONTROL
Plate assay
Thermostatic control required in microbial assay during culturing , inoculam
preparation and incubation
Tube assay
Close control of temperature is required. Circulated air or water may be used.
Water is more advantage because of greater heat capacity

27. CYLINDER PLATE METHOD
One level assay with standard curve
Two level factorial assay
One level assay with standard curve
Standard solution (stock solution) – Five test dilutions are prepared
Sample solution – prepare dilution to a concentration equal to median level of standard to
give sample solution
Method
Prepare the standard curve using 12 petri dishes to accommodate 72 cylinders
or cavities. Fill the cavities with solution (test dilution)S3 representing median
concentration of the standard solution. Incubate and estimate the potency

28. Estimate the potency
Formula L = (3a + 2b + c – e) / 5 H = (3e + 2d + c – a) / 5
L = calculated zone diameter for lowest concentration of the standard curve
H = calculated zone diameter for highest concentration of the standard curve
C = average zone of the 36 readings of the reference point standard dilution
a,b,d,e = average value of other standard solution (lowest and highest concentration)
Two level factorial assay
Parallel dilutions containing two levels of both standard (S1 + S2) and the unknown
(U1 + U2) % Potency = antilog (2.0 + a.log I)
Where a = {(U1 + U2) - (S1 + S2)} / {(U1 + U2) +(S1 + S2)}
(U1 + U2) = sums of the zone diameters with unknown solutions of high and low concentrations
(S1 + S2) = sums of the zone diameters with standard solutions of high and low concentrations
I = ratio of dilutions
Potency of sample = (% potency X assumed potency of sample) / 100

29. Turbidometric or tube assay method
Advantage – Short incubation method
Disadvantage – prepare of solution residue and inhibition substances
Estimation of potency
L = (3a + 2b + c – e) / 5 H = (3e + 2d + c – a) / 5
Where ,
L = calculated absorbance(lowest concentration of the standard curve)
H = calculated absorbance(highest concentration of the standard curve)
Precision of microbiological assay
Minimum acceptable – < 95 % and > 105 % of estimated potency unless stated in monograph
Incase of potency stated on label (antibiotic preparation) < 98 % and > 102 %
By adding this Fiducial limits and the final preparation failing to comply with the
official requirements of potency can be overcome.

30. • The principle of microbiological bioassays for growth factors such as vitamins and
amino acids is quite simple.
• Unlike antibiotic assays which are based on studies of growth inhibition.
• These assays are based on growth exhibition.
• All that is required is a culture medium which is nutritionally adequate for the test
microorganism in all essential growth factors except one being assayed.
• If a range of limiting concentration of the test substance is added, the growth of the
test organism will be proportional to the amount added.
• A calibration curve of concentration of substance being assayed against same
parameters of microbial growth.
Eg: cell dry weight, optical density or acid production can be plotted.
Microbial assay of Vitamins

31. Standard curve of vitamin assay
 From the standard curve, the concentration of growth factor in the unknown solution can
be determined.
 It is possible to assay a variety of different growth factors with a single test organism
simply by preparing a basal media with different growth limiting nutrients.
 Lactic acid producers are the micro organisms of choice because of their advantages
characteristics.
Vitamin Concentration in mg
Optical density

32. S.No Assay micro organism Vitamins
1 Lacto bacillus Biotin
2 L. arabinosus Calcium pantothenate
3 L.leichmannji Cyanocobalamine
4 L. casei. Folic acid
5 Streptococcus faecalis Folic acid
6 L.arabinosus (or)L.platarum Nicotinic acid
7 L.casei Riboflavin
8 L.viridans Thiamine
9 L.casei Pyridoxal
10 Neurospora crassa / Sacchromyces
grisebergensis
Pyridoxine
11 Acetobactor suboxydans Pantothenal
12 Sacchromyces uvaram Inositol

33. • They are better lactic acid producers, therefore they are considered to be more
suitable for acidimetric methods involved in the measurement of growth response.
• Usually titration of lactic acid is done against 0.1N NaOH using Bromothymol as the
indicator.
• They posses iniform biochemical characteristics compared to many bacteria such as
E.coli, Lactobacillus bacteria are extremely stable and apparently have a very low
mutation rate.
• All the lactic acid bacteria have relatively complex and exact nutritional
requirements.
• None of the Lactobacillus of leuconostoc species are known to be pathogenic/ to
cause food poisoning in man.
Advantage of lactic acid producing organisms

34. • Suspend 0.200 g in 5 ml of dimethylformamide R. Heat until the substance has
dissolved completely.
• Add 50 ml of ethanol R and titrate with 0.1 M tetrabutylammonium hydroxide,
determining the end-point potentiometrically
 Apply to the plate 10 µl of each solution.
 Develop over a path of 15 cm using a mixture of 5 volumes of methanol R, 25 volumes
of glacial acetic acid R and 75 volumes of toluene R.
 Dry the plate in a current of warm air.
 Allow to cool and spray with 4-dimethylaminocinnamaldehyde solution R.
 Examine immediately in daylight.
 Any spot in the chromatogram obtained with test solution
Method
ASSAY