The document discusses the production of antibiotics and antitumor agents through industrial microbiology. It defines antibiotics as substances produced by microorganisms that inhibit or kill other microorganisms. Antibiotics are produced through the fermentation of microorganisms like Streptomyces. The production process involves growing the culture in large tanks, isolating the antibiotic, and purifying it into final products through various chemical processes. Quality control ensures antibiotics meet standards before distribution. Some antibiotics like anthracyclines also have antitumor properties and are used to treat cancer.

1. SCHOOL OF BIOSCIENCE AND TECHNOLOGY
DEPARTMENT OF BIOLOGY
Production of Antibiotics and Antitumor Agents
Seminar on the Course of Industrial Microbiology (BIOLM)
By: Gedefaw Wubie
June, 2019
Dessie; Ethiopia
1

2. Outline of the presentation
 Key terms
 Definition of antibiotics
 Production of antibiotics
 Uses of antibiotics
 Classification
 Drug susceptibility testing
• Classification of antibiotics
2

3. Key terms and phrases
– Chemotherapy
• The use of drugs to treat a disease
– Antimicrobial drug
• A chemical that destroys pathogens without damaging
body tissues
– Antibiotic
• A substance produced by microorganisms, that in small
amount, inhibits another microorganisms
– Chemotherapeutic agent
• A synthetic chemical (drug) used in the treatment of
disease
3

4. Meaning of antibiotics
 Literal translation
– anti – against
– biotic – living things
 It is a chemical substance
produced by a microorganism
that inhibits the growth of or kills other microorganisms.
 The term antibiotic was coined from the word antibiosis which
literally means against life.
 It defined as a substance, produced by one microorganism
(Denyer et al., 2004), or of biological origin (Schlegel, 2003)
which at low concentrations can inhibit the growth of, or are
lethal to other microorganisms (Russell, 2004).
4

5. The history of antibiotics
 1928 – Alexander Fleming discovered
penicillin, produced by Penicillium
 The early work done by
Fleming, who discovered the
effect of penicillin
 Development of this and other
antibiotics such as actinomycin
and aureomycin and other
tetracyclines solved many
problems during the second
world war.
 1940 – Howard Florey and Ernst
Chain performed 1st clinical trials of
penicillin
5

6. History of chemotherapy
– Paul Ehrlich
• In the early 20th century, while
attempting to stain bacteria
without staining the surrounding
tissue, he speculated about
“Magic bullet”
– That would selectively find and destroy pathogens but not
harm the host
• The father of chemotherapy
• With Sahachiro Hata developed Salvarsan
(Arsphenamine) against syphilis in 1910
6

7. Sources of Antibiotics
 Natural microbial production using fermentation technology.
Example: Penicillin
 Semi synthetic production (post production modification of
natural antibiotics). Example: Ampicillin
 Synthetic production of antibiotics made synthetically in the
lab. Example: Quinoline
7

8. Cont…
– More than half of our antibiotics are produced by species of
Streptomyces, filamentous bacteria that commonly inhibit
soil
– It is interesting to note that practically all antibiotic
producing microbes have some sort of sporulation process
8

9. Table 20.1 Representative sources of antibiotics
9
Microorganism Antibiotic
Gram + rods
Bacillus subtilis Bacitracin
Bacillus polymyxa Polymyxin
Actinomycetes
Streptomyces nodosus Amphotericin B
S. venezuelae Chloramphenicol
S. aureofaciens Chlortetracycline & tetracycline
S. erythraeus Erythromycin
S. fradiae Neomycin
S. griseus Streptomycin
Micromonospora purpureae Gentamicin
Fungi
Cephalosporium spp. Cephalotin
Penicillium griseofulvum Griseofulvin
P. notatum Penicillin

10. Antibiotics production
 Microbial production of antibiotics by secondary metabolisms
 Because the production of antibiotics is non-growth
associated, using cell immobilization to uncouple cell growth
and metabolite production is an effective method of improving
the process.
10

11. Steps in the Production of Antibiotics
 It is long and a basic research proposal with d/t
manufacturing requirements
 1st species are screened for any sign of antibacterial
action.
 The species is tested against a variety of known infectious
bacteria.
 the organism is grown on a large scale so the compound
responsible for the antibiotic effect can be isolated.
 Go to clinical testing to prove that the antibiotic works in
animals and humans and is not harmful.
 If these tests are passed, the (FDA) must then approve the
antibiotic as a new drug. This whole process can take
many years (Stinson and Stephen, 1996).
11

12. Raw Materials
 fermentation broth are the primary raw materials required for
antibiotic production.
 This broth is an aqueous solution made up of all of the ingredients
necessary for the proliferation of the microorganisms.
 Typically, it contains a carbon source, like molasses, or soy meal,
both of which are made up of lactose and glucose sugars.
 These materials are needed as a food source for the
organisms. Nitrogen is another necessary compound in the
metabolic cycles of the organisms.
 phosphorus, sulfur, magnesium, zinc, iron, and copper introduced
through water soluble salts. Anti-foaming agents such as lard oil,
octadecanol, and silicones are used. 12

13. The Manufacture Process
 It involves
Isolating a desired microorganism
fueling growth of the culture and
refining and isolating the final
antibiotic product.
 Sterile conditions must throughout the
manufacturing process
 If no, there is contamination by
foreign microbes will ruin the
fermentation.
13

14. Starting the Culture
Before fermentation, the desired antibiotic-producing organism must be
isolated and its numbers must be increased by many times.
a starter culture from a sample of previously isolated, cold-stored
organisms is created in the lab.
In order to grow the initial culture, a sample of the organism is
transferred to an agar-containing plate.
The initial culture is then put into shake flasks along with food and
other nutrients necessary for growth. This creates a suspension, which
can be transferred to seed tanks for further growth.
14

15. Fermentation
 MOs are allowed to grow and multiply.
 they excrete large quantities of the desired antibiotic.
 The tanks are cooled to keep the temperature between 73-81°
F (23-27.2 ° C).
 It is constantly agitated, and a continuous stream of sterilized
air is pumped into it.
 For this reason, anti-foaming agents are periodically added.
Since pH control is vital for optimal growth, acids or bases are
added to the tank as necessary.
15

16. Isolation and purification
 After 3to 5days, the maximum amount of antibiotic will have
been produced and the isolation process can begin.
 Depending on the specific antibiotic produced, the
fermentation broth is processed by various purification
methods.
 The dissolved antibiotic is then recovered using various
organic chemical means.
 At the end of this step, the manufacturer is typically left with a
purified powdered form of the antibiotic, which can be further
refined into different product types.
16

17. Refining
 Antibiotic products can take on many different forms. They
can be sold in solutions for
– intravenous bags or syringes,
– in pill or gel capsule form,
– powders, which are incorporated into topical
ointments.
 Depending on the final form of the antibiotic, various refining
steps may be taken after the initial isolation.
 the antibiotic product is then
Packed and
transported to various distributors,
hospitals, and pharmacies.
 The entire process of fermentation, recovery, and processing
can take anywhere from five to eight days.
17

18. Quality Control
• No contamination at any point during production.
• Medium + all of the processing equipment = steam sterilized.
• Physical + chemical properties of the finished product are checked such as
• pH
• MP and
• MC (Crueger, 1998).
• FDA requires that for certain antibiotics:
– Must be checked the effectiveness and purity.
– Only after they have certified the batch can it be sold for general
consumption.
• In fact the development of a new drug is a costly proposition, However, an
alarming development has encouraged a re-energized interest in the
development of new antibiotics.
18

19. Therapeutic index must be calculated
– The ratio between toxic dose and therapeutic dose
– High therapeutic index
 less toxic or the safer the drug
 High therapeutic dose less toxic dose is safer
19

20. Antitumor Antibiotics
• Antitumor antibiotics (cytotoxic/anticancer antibiotics) are
drugs that inhibit and combat the development of tumors.
• Anthracyclines are important group of antitumor antibiotics
and seven members of this group have been shown to be
clinically important in cancer treatment which include:
» daunorubicin
» doxorubicin
» epirubicin
» idarubicin
» pirarubicin
» zorubicin and aclarubicin (Fischer et al., 2003).
20

21. Cont…
• Anthracycline first isolated as red substances from
microorganisms in 1939 and their antibiotic properties were
studied in the 1950s.
• These antibiotics killed bacteria quite readily but were too toxic to
be used against the infections in humans.
• It was after 1960s that anthracycline antibiotics were tested for
antitumor properties and found to be active against cancer cells
(Taatjes et al., 1997).
• Among all groups of microorganisms, the antitumor antibiotics
produced by Streptomyces are invaluable in the medical field
(Mueller and Nicole 2002).
• All the seven members of anthracyclines are produced by
Streptomyces species (Martins and Souto- Maior, 2003).
21

22. Cont…
• Mostly produced by staged fermentations where the source microorganism is
grown in large containers containing a liquid growth medium (Madigan and
Martinko, 2005).
• Batch fermentation used for the production of anthracyclines (Ciclamycin) from
Streptomyces capoamus (Martins and Souto- Maior, 2003).
• Industrial production of daunorubicin and doxorubicin has also been reported
(White and Stroshane, 1984) but very little has been published on process and
media development for maximal titre production of anthracyclines in commercial
fermentations (Martins and Souto-Maior, 2003).
• Increasing the shaking speed and decreasing the medium volume improves
antitumor production.
• HIGH glucose uses in catabolite repression of the biosynthesis of the antitumor
antibiotic.
• Also increasing antibiotic activity was observed both intra and extracellularly
during growth under the carbon and nitrogen limiting conditions (Lilley et al.,
1981).
22

23. Role of Antibiotics
 Bacteriostatic = To inhibit multiplication
 Inhibit bacterial growth
rely on host immunity
 Bacteriocidal = Kill bacteria
– Most useful in situations when host defenses cannot control
pathogen
» (walsh, 2003)
23

24. Cont…
Bacteriostatic antibiotics
 Tetracyclines
 Spectinomycin
 Sulphonamides
 Macrolides
 Chloramphenicol
 Trimethoprim
Bactericidal antibiotics
 Penicillins
 Cephalosporins
 Fluoroquinolones (Ciprofloxacin)
 Glycopeptides (Vancomycin)
 Monobactams
 Carbapenems
24

25. Spectrum of activity
• For antibiotic to be an effective drug:
– It must cause a significantly greater harm to the pathogen than to the
host being treated (selective toxicity)
Narrow spectrum
– Work on narrow range of organisms
» Gram+VE only OR Gram-VE only
– Advantage: effects pathogen only
– Disadvantage: requires identification of pathogen
Broad spectrum
– Advantage: Work on broad range of organisms
– Disadvantage : disruption of normal flora
– This require the targeting of features of the pathogen that differ from
the host’s cells
• Bacteria have several such targets such as cell wall and ribosomes
• Viruses and eukaryotic pathogens are much more challenging to
treat since they have less features that can be safely targeted
25

26. Antibiotic Classes
Penicillins
penicillin
amoxicillin
Cephalosporins
cephalexin(Keflex)
Sulfonamides
co-trimoxazole (Bactrim)
Fluoroquinolones
ciprofolxacin (Cipro)
levofloxacin (Levaquin)
ofloxacin (Floxin)
Tetracyclines
Tetracycline(Sumycin)
doxycycline (Vibramycin)
Aminoglycosides
gentamicin (Garamycin)
kanamycin
tobramycin(Tobrex)
Macrolides
erythromycin (E-Mycin)
azithromycin (Zithromax)
Prevents
bacteria from
making cell
walls
Inhibits
DNA
replication
Inhibits
protein
synthesis
Inhibits
Folate
synthesis

27. The major modes of action of antimicrobial drugs
27

28. 28

29. Inhibition of Cell Wall Synthesis
– Bacterial cell wall is made of peptidoglycan
– PG is found only in bacteria
– Human cell don’t have peptidoglycan cell wall
– Since they affects cell wall synthesis process
• Only actively growing cells are affected
29

30. 30
Figure:The structure of penicillins

31. 31
Figure 20.8 The effect of penicillinase on penicillins

32. Inhibition of Protein Synthesis
– Protein synthesis is a common feature for all cells
– But there is difference in ribosomes structure
• Prokaryotes have 70S ribosomes (30S + 50S)
• Eukaryotes have 80S ribosomes (40S + 60S)
– “S” for Svedberg unit, the relative rate of sedimentation in a
high speed centrifuge
– Mitochondria – important eukaryotic organelles also contain
70S ribosomes similar to prokaryotes
» Therefore, antibiotics targeting the 70S can have adverse
effect on the host cells
32

33. 33
Figure 20.4 The inhibition of protein synthesis by antibiotics

34. 34

35. Cont…
– Most drugs that inhibit protein synthesis have a broad
spectrum of activity
• Chloramphenicol
• Aminoglycosides
– Streptomycin, neomycin, gentamycin
• Tetracyclines
• Erythromycin is an exception
– Since it doesn’t penetrate gram negative cell wall
35

36. Injury to the Plasma Membrane
– Especially polypeptide antibiotics
• Cause change in the permeability of the plasma membrane
– Polymyxin B
• Cause disruption of the plasma membrane by attaching to the
phospholipids of the membrane
– Against gram negative bacteria
– Amphotericin B, miconazole, ketoconazole
• Antifungal drugs
• By combining with sterols … disrupts fungal plasma
membrane
• Don’t act on bacteria since bacteria lack sterol
• Can be toxic to host cell since animal cell have sterols
36

37. • Animal cell have mostly cholesterol
Vs
• Fungal cell have mostly ergosterol against
which the drug is most effective
– So that the balance of toxicity is tilted against the
fungus
37
Figure. Injury to the plasma
membrane of a yeast cell
caused by an antifungal
drug, miconazole

38. Inhibition of Nucleic Acid Synthesis
– Rifampin and quinolones
• Interfere with the process of DNA replication and
transcription in microorganisms
• More selectively toxic
– Rifampin
• Inhibit synthesis of mRNA
• Treatment for tuberculosis
– Quinolones
• Inhibit DNA synthesis
• Broad spectrum
• Treatment for urinary tract infections
38

39. Inhibition of the Synthesis of Essential
Metabolites
– Para-aminobenzoic acid (PABA)
• Substrate for an enzymatic reaction leading to the synthesis
of folic acid
• Folic acid
– A vitamin that functions as a coenzyme for the synthesis
of nucleic acids and many amino-acids
– Important for the growth of the microorganism
– Sulfanilamide
• Competitor to PABA
• In its presence, the enzyme combine with sulfanilamide in
steady of PABA … which prevents folic acid formation
39

40. • Human don’t produce folic acid as a result
sulfanilamide selectively affects microorganisms that
synthesize their own folic acid
40
Figure 5.7 Enzyme Inhibitors

41. Antimicrobial Sensitivity Test
– Necessary only:
• When susceptibility of a pathogen is not predictable or
• When antibiotic drug resistance problems develop
• The Diffusion Methods
– The most widely used method
– The method of testing is disc-diffusion method
• Also known as the Kirby-Bauer test
41

42.  Results reported as:
– Sensitive
– Intermediate
– Resistant
42
Figure: The disc-diffusion method for determining the
activity of antimicrobials
• Results are often inadequate for many clinical purposes
• The test is simple and inexpensive
• Mostly used when more sophisticated lab facilities are not available

43. – E-test
• A more advanced diffusion method
• Enable to estimate the minimal inhibitory concentration
(MIC)
– The lowest antibiotic concentration that prevents visible
bacterial growth
43
Figure 20.18 The E test (for
epsilometer), a gradient
diffusion method that
determines antibiotic
sensitivity and estimates
MIC

44. E-test
• A more advanced diffusion method
• Enable to estimate the minimal inhibitory
concentration (MIC)
– The lowest antibiotic concentration
that prevents visible bacterial growth
44
Figure : The E test (for
epsilometer), a gradient
diffusion method that
determines antibiotic
sensitivity and estimates
MIC

45. The Broth Dilution Tests
– A weakness of the diffusion method is that it doesn’t
determine whether a drug is:
• Bactericidal or bacteriostatic
– But this method determine
• MIC
– By making a sequence of decreasing concentration
• Minimal bactericidal concentration (MBC)
– By culturing wells that don’t show growth or higher
concentration than the MIC in drug free broth
45

46. 46
Figure 20.19 A microdilution or microtiter, plate used for testing for MIC of antibiotics

47. Antibiotic Resistance
 Meaning:
 The ability of a microorganism to survive at a given
concentration of an antimicrobial agent at which the normal
population of the microorganism would be killed this is called
the “Epidemiological breakpoint”.
47

48. cont…
A variety of mutations can lead to resistance
– Mechanisms of antibiotic resistance:
48

49. 49

50. Emerging problems
– Fluoroquinolones-resistant Salmonella – 3rd gen.
– Cephalosporin-resistant Salmonella– Fluoroquinolone- and
– macrolide-resistant Campylobacter
– Vancomycin resistant enterococci (VRE) – (Multiresistant E.
coli)
–
50

51. Cont…
– Evolution of drug resistance could be
• Vertical … due to spontaneous mutation
• Horizontal … due to gene transfer e.g. gene in plasmids
– Misuse of antibiotics selects resistance mutants
– Misuse includes:
• Using outdated or weakened antibiotics
• Using antibiotics for the common cold and other inappropriate
conditions
• Using antibiotics in animal feed
• Failing complete the prescribed regimen
• Using someone else's leftover prescription
51

52. Effect of combination of drugs
 Combination sometimes used to treat infections
Synergism (Synergistic: whole is > sum)
• When the effect of two drugs together is greater than the
effect of either alone
– E.g. penicillin and streptomycin for bacterial endocarditis
Antagonism (Antagonistic: whole is < sum)
• When the effect of two drugs together is less than the effect
of either alone
– E.g. penicillin and tetracycline
Additive: whole is the sum
52

53. Antibiotic resistance crisis
An increase in resistant organisms
+
A limited number of new antimicrobial drugs
=
a problematic scenario
“The pharmaceutical industry has largely turned away from antibiotic research due to the low
likelihood of getting a return on investment. Any new class of antibiotics would need to be used
sparingly to conserve their effectiveness, meaning sales would be slow.” - WSJ

54. Post-antibiotic era
Currently:
- 80% of gonorrhoeal infections are now resistant to antibiotics.
- 440,000 new cases of drug-resistant tuberculosis annually.
Sally Davies (Britain’s Chief Medical Officer)
“Antibiotic resistance should be added to the list of
national emergencies”.
In the future:
- - The cost to treat drug-resistant cases is estimated to be at
least double.

55. 55

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