STREPTOMYCIN

STREPTOMYCIN--IT'S HISTORY,DISCOVERY,PRODUCTION,USES AND BIOSYNTHESIS

1. Streptomycin: ---It’s
history,production,
BIOSYNTHESIS and importance--
-treatment of various diseases
PREPARED BY
DEBANGANA MOITRA

2. Evening of March 24,1882–Robert Koch –German physician and
scientist--presented his discovery of Mycobacteriumtuberculosis, that
causes tuberculosis(TB)---“one seventh of all human beings die of
tuberculosis and…if one considers only the productive middle-age
groups, tuberculosis carries one-third and often more of these…”
Despite Koch’s discovery, for 60 years the only treatment of
tuberculosis was isolation, “fresh air, and sunshine”

3. So it was time for another Nobel laureate!
Selman Waksman
1888-1973
A childhood immigrant
from the Ukraine.
A Russian-born
American
microbiologist.
Through hard work, he
became a professor of
microbiology and
biochemistry at
Rutgers.

4. The mass manufacture of penicillin during World War II
stimulated urgent interest in other medicinally important soil
microorganisms.
Waksman, had been engaged in a study of soil microbes for a
number of years.
One of his students, French-born RenéJules Dubos (1901-
1981), was searching for antibacterial substances in soil.
In 1939 Dubos discovered the first antibiotic drug, gramicidin.
Although it fought pneumococcus, staphylococcus, and
streptococcus bacteria, it was too toxic (poisonous) for use in
humans.

5. Waksman was inspired by Dubos's discovery.
With the support of the Merck pharmaceutical company, he turned his
attention to antibacterial substances found in soil.
In 1941, he dubbed these substances "antibiotics.”
By fall 1943, Waksman had his lab group concentrating on the
Actinomyceteswhen William Feldman and Corwin Hinshaw, visiting from
the Mayo Clinic, presented a clinical trial opportunity for an anti-
tuberculosis drug.
Waksman assigned preparation for that task to his graduate students,
chiefly Albert Schatz.
Schatz soon isolated streptomycin, an aminoglycoside that inhibits
protein synthesisand found to be active against gram negative bacteria.

6. Streptomycin
Streptomyces griseus.
Isolated on October 19, 1943by Albert Schatz,
a graduate student in Waksman’s lab

7. WHAT IS ANTIBIOTIC?
•Antibiotic, also called antibacterial, is a type
of antimicrobial drug.
•Used in the treatment and prevention of
bacterial infections.
•They may either kill or inhibit the growth of
bacteria.
•They are derived from bacterial
sources(microorganisms) or plant sources.

8. STRUCTURE
•Streptomycin is characterized chemically as an aminoglycoside
antibiotic. It has the chemical formula C21H39N7O12. It
consists of three components linked glycosidically (by ether
bonds):
•(i) Streptidine (inositol with two guanido groups),
•(ii) Streptose (methyl pentose), and (iii) Streptoscamine (N-methyl-L-
glycosamine) as shown in Fig. 45.9.
•Both guanido groups of streptidine are essential for the antibiotic
activity and removal of one group reduces antibiotic activity up to 90%.

9. PROPERTIES OF STREPTOMYCIN
•Streptomycin is soluble in water but insoluble in acetone, chloroform, and
ether.
•Streptomycin is relatively stable. According to Regna, Wassele, and
Solomon, there was no loss of potency in commercial samples that were
stored at room temperature for 12 months containing less than 1% of
moisture.
•Strong alkaline solutions are destructive to streptomycin, the action being
very rapid when the solutions are boiled.
•Streptomycin is highly resistant to the action of enzymes and other
biological agents.
•Streptomycin is relatively nontoxic to man. But after the use of
streptomycin, reactions such as presence of pain, irritation, headache,
fever, blood pressure drop, disturbances in eighth cranial nerve, skin
eruption, albumin in urine may take place.

10. Streptomycin kills many bacteria, including
Mycobacterium Tuberculosis!
As the tubercle bacillus can enclose itself in
nodules in the body, it can remain dormant for
years. When TB becomes active, it can strike
any part of the body, but normally attacks the
lungs
BUT HOW?
Bugs fight bugs

11. The central dogma--
anybody remember that?
DNA -> RNA -> Proteins

12. And we call that step?
DNA -> RNA -> Proteins

13. Translation!
DNA -> RNA -> Proteins

14. Bacteria do it too,
and like us they use RIBOSOMES

15. The ribosome is an amazing machine
That unlike most in the cell runs on RNA!
16S rRNA + proteins =30S or small subunit

16. MECHANISM OF ACTION
•Streptomycin is aprotein synthesis inhibitor. It binds to the small 16S
rRNA of the 30S subunit of the bacterial ribosome, interfering with the
binding offormyl-methionyl-tRNAto the 30S subunit.
•This leads to codon misreading, eventual inhibition of protein synthesis
and ultimately death of microbial cells through mechanisms that are still
not understood.
•Speculation on this mechanism indicates that the binding of the
molecule to the 30S subunit interferes with 50S subunit association with
themRNAstrand.
•This results in an unstable ribosomal-mRNA complex, leading to
aframe shift mutationand defective protein synthesis; leading to cell
death.

17. MECHANISM OF ACTION
•Humans have ribosomes which are structurally different from those in
bacteria, so the drug does not have this effect in human cells.
•At low concentrations, however, streptomycin only inhibits growth of the
bacteria by inducing prokaryotic ribosomes to misread mRNA.
•Streptomycin is an antibiotic that inhibits both Gram-positive and Gram-
negative bacteria,and is therefore a useful broad-spectrum antibiotic

18. MECHANISM OF ACTION
•Streptomycin causes a structural change which interferes with the
recognition site of codon-anticodon interaction resulting in misreading of
the genetic message carried by messenger RNA (mRNA). The
mechanism of inhibition of protein synthesis by streptomycin is shown as
follows:--

19. If we zoom in further…

20. Okay. So, streptomycin kills bugs in
a flask in the lab. What about
inside a patient?

21. Now we need to try it on animals.
Enter pathologist Dr. William Hugh Feldman and bacteriologist
Dr. H. Corwin Hinshaw
at the Mayo Clinic
In two months they reported to Waksman
that four tubercular guinea pigs receiving streptomycin
"look exceedingly well."
We do, don’t we!

22. Injection of streptomycin to guinea pigs

23. OK, but how about people?
NextFeldman and Hinshawinvent clinical trials

24. OK, but how about people?
In August 1945 Hinshaw reported that
thirty-three patients had been treated
"and [we] continue to be quite optimistic”.
Streptomycin
appeared to be a miracle cure
•AfterFeldman and Hinshaw carried out animal testing ,they treated
"Patricia T." between November 1944 and February 1945. She was
streptomycin's first success against TB.

25. •At the end of World War II, the United States Army experimented with
streptomycin to treat life-threatening infections at a military hospital
inBattle Creek, Michigan. The first patient treated did not survive; the
second patient survived but became blind as a side effect of the treatment.
In March 1946, the third patient—Robert J. Dole, later Majority Leader of
the United States Senate and Presidential nominee—experienced a rapid
and robust recovery.
[24]

26. •The first randomized trial of streptomycin against pulmonary tuberculosis
was carried out in 1946 through 1948 by the MRC Tuberculosis
Research Unit under the chairmanship of Geoffrey Marshall (1887–
1982). The trial was bothdouble-blindandplacebo-controlled. It is widely
accepted to have been the first randomized curative trial.
•Results showed efficacy against TB, albeit with minor toxicity and
acquired bacterialresistanceto the drug.
[26]

27. Now, we have to make a lot of it.
In steps George Merck
of Merck and Co.

28. Now, we have to make a lot of it.
In steps George Merck
of Merck and Co.
And we got the patent…
THE US PATENT OFFICE ISSUED A PATENT (NO. 2,449,866; SEPT. 21, 1 948) TO
WAKSMAN AND SCHATZ FOR "STREPTOMYCIN AND PROCESS OF
PREPARATION," WHICH THEY ASSIGNED TO THE RUTGERS RESEARCH AND
ENDOWMENT FOUNDATION. COMMERCIAL DEVELOPMENT .

29. ORGANISMS USED : --
The organisms used for streptomycin
production are strains of
Streptomyces griseus, an
actinomycetes that produces aerial
mycelium and spores. The
streptomycin-producing qualities of
Streptomycesgriseushave been
improved by strain selection and by
irradiation with ultraviolet light,
according to Stanley.

30. MATERIALS AND METHODS
•Instruments: ---Laminar air flow, Autoclave, Hot air oven, Shaker
incubator, Microscope, Cooling centrifuge, HPLC.
•Sample collection: ---The soil sample was collected from Chamundihill
at Mysore city.
•Isolation and identification of S. griseus: ---The strain of S. griseus was
isolated form soil sample by using serial dilution technique and spread
plate method with Starch casein agar medium. S. griseus was identified
by following tests:
•Gram staining, Starch hydrolysis, Casein hydrolysis, Fermentation of
carbohydrate (glucose fermentation), Hydrogen sulfide (H2S) production,
Urease production, Methyl-Red and Voges-Proskauer(MRVP), Indole
production, Citrate utilization, Hydrolysis of gelatin and Catalase test.

31. MEDIA COMPOSITION: -
•Following is the
composition of medium
in 1000 ml distilled
water: --
Soya bean
meal
10 gm.
Glucose10 gm.
Peptone5 gm.
Meat
extract
5 gm.
Sodium
chloride
5 gm.

32. MEDIA COMPOSITION: --
•The pH is kept at 7.6-8.0 before sterilization and after
inoculation the culture is incubated at 28 C.
•Bennett reported that the following raw materials
gave comparable yields of streptomycin on a laboratory
scale: corn-steep water, soya bean flour, some
peptones, acid hydrolyzed rabbit fur, acid hydrolyzed
wheat gluten, acid hydrolyzed stillage obtained from
the yeast-alcohol fermentation of wheat mash, and
asparagus butt juice.

33. COMMERCIALPRODUCTION: ---
HISTORY: --The first concern to manufacture
streptomycin industrially was Merck and Company,
which was in operation by the spring of 1946.
Streptomycin hydrochloride and streptomycin
sulphate were produced initially but they could not
be as highly purified as was desired. In 1945, Peck
and coworkers reported the preparation of a
crystalline double salt of streptomycin
trihydrochloride and calcium chloride which was of
high purity. This salt has the formula
C21H39O12N7.3HCL----1/2 CaCl2. Since July 1947,
Merck and Company has manufactured the
crystalline calcium chloride salt streptomycin
exclusively.
Crystalline streptomycin
trihydrochloride calcium chloride
double salt (Merck & Co.)

34. 1.The raw materials are mixed with water in a mixing tank at about
10 times the concentration in which they will be used and pumped
to a storage tank.
2. From the storage tank, the concentration is pumped
into each of a series of four fermenters of increasing sizes as
required.
3. The fermented medium, diluted with water to the desired
concentration, is then sterilized at 120 C., cooled and inoculated
with a starter.
FERMENTATION

35. Partial view of fermentation unit in plant of Merck & Co., Inc., at Elkton,
U.S.

36. FERMENTATION
4. The fermentation is carried out at 25 to 30 C, with aeration
and agitation for the required length of time, the contents of
each fermenter serving as the inoculum for the next larger
fermenter.
5. Aeration is supplied by blowing sterile air through the
fermentation medium and by agitation. The rate of aeration
and agitation must be controlled to prevent foaming. Antifoam
agents may be used if excessive foaming occurs.

37. FERMENTATION
6. There are three main steps in the production process of
streptomycin: ---
First Phase: --Growth of mycelium occurs, the proteolytic activity
of S.griseusreleases ammonia from the soya bean meal, the
carbon from soya bean meal utilizes and induce growth but
glucose is utilized at a minimum rate. The yield of streptomycin
produced is low.
Second phase: --The streptomycin synthesized at a rapid rate in
this phase, due to rapid utilization of ammonia and glucose. The
total incubation period lasts from 24 hours to 6-7 days. No
mycelial growth occurs in this phase. The pH remains 7.8 to 8.

38. FERMENTATION
•Third phase: --The sugar depletes from the medium
resulting into cease of streptomycin production. The
cells lyse, releasing ammonia resulting into raised pH.
Before lysis, fermentative material is harvested.
7. The fermentation, which is carried out at 25 to 30 C.,
is considered completed when a maximum yield of
streptomycin has been obtained.

39. FILTRATION: ---
The fermented broth is
filtered to remove the
mycelium. This is
accomplished in Oliver
precoated pressure filters,
which have high filtration
rate. The steps are as
follows---
Before the broth goes to
the filter, filter aid (non-
adsorbing) is mixed with
it in a slurry mixing tank,
the amount added
depending upon the
rate of flow of liquor.

40. FILTRATION
•The admixture flows through the filter which has been precoated with
filter aid.
•After this primary filtration, the pH of the filtrate is adjusted.
•Then it is polished by passage through a second filter. The filter cakes
are discarded.

41. ADSORPTION OF ACTIVATED
CARBON: --
•Activated carbon is mixed with the clear broth in a
series of three adsorption tanks.
•The streptomycin and some of the impurities are
extracted from the broth by adsorption on the
activated carbon.
•It is then automatically fed to a pressure filter, where
the spent broth is separated out.
•The adsorbate is washed on the filter with dilute
alcohol for the purpose of removing impurities soluble
in alcohol.

42. ELUTION
The streptomycin is eluted from the activated carbon
with dilute alcoholic hydrochloric acid by a two-stage
countercurrent process.
It is removed as the trihydrochloride, while impurities
remain adsorbed on the activated carbon.
The trihydrochloride is separated from the activated
carbon by filtration.
The elution units are especially constructed in
order to prevent the formation of metallic salts that
would contaminate the streptomycin. They are
composed of elution tanks lined with rubber, pipes
and fittings of porcelain.

43. CONCENTRATION
•The acid eluate is neutralized
and then concentrated with
single-phase evaporators. It is
passed through a series of
three evaporators, which are
maintained under vacuum at
60 C. The concentrate leaving
the third evaporator or
dehydrator contains
approximately 25% of solids of
which less than ¼ th is
streptomycin, according to
Porter.

44. SOLVENT PRECIPITATION
•The concentrate thus obtained is treated with acetone which precipitates
the streptomycin hydrochloride.
•The mixture is filtered through a plate-and-frame press, the filtrate going
to a solvent recovery unit.
•The precipitated streptomycin is redissolved in alcohol and mixed with an
adsorbing material to remove only the impurities present in streptomycin,
but not the streptomycin from solution. The mixture is filtered.

45. •FORMATION OF COMPLEX: --
•An alcoholic solution of calcium chloride is
added to an anhydrous alcoholic solution of
streptomycin. The crystalline complex is of
high purity (98% or higher).
•FINAL OPERATIONS: ---
Under aseptic conditions, the crystalline
complex is dissolved in pyrogen-free
water.
It is then passed through a biological
filter to remove any microorganisms.
Then, it is dried in stainless-steel trays
from the frozen state under vacuum.

46. Now, the dried cake is powdered
from high vacuum sublimation, using
sterile steel balls.

47. Next, the streptomycin powder is weighed into sterile vials. This procedure is carried
out on delicate prescription balances in sterile cubicles. The entire room is air-
conditioned with filtered air and “sterility” is maintained through the use of ultraviolet
ray lamps.

48. Next, it is packaged. The last operations are carried out in rooms maintained at 10%
relative humidity.
1948 STREPTOMYCIN PRODUCTION VIDEO

49. PREVENTION OF CONTAMINATION: --
To prevent contamination of the product, the workers must use special cleaning
techniques and change into sterile uniforms and shoes before going to the processing
areas.
In order to reduce the numbers of microorganisms in the air, all the air entering the
building is passed through special filters.
Triethylene glycol is used as a disinfecting aerosol.
Ultraviolet lamps are used in the cubicles where the antibiotic is exposed to the air for
a short time.

50. STANDARDIZATION
Waksman proposed the establishment of the following units: -
1.An S unit, or that amount of material which will inhibit the growth of a
standard strain of E.coli in 1 ml of nutrient broth or other suitable
medium. This unit would thus correspond to the original E.coli unit.
2. An L unit, or that amount of material which will inhibit the growth of a
standard train of E.coli in 1 liter of medium. An Lunit is thus equivalent to
1000 S units.
3. A G unit comparable to one gram of the crystalline material.

51. METHODS OF ASSAY
Biological and chemical methods of assay have been employed but the
biological methods are the most suitable at the present time.
BIOLOGICAL METHODS : --
•Cup method of Stebbins and Robinson.
•Paper disc method of Loo and associates.
•Broth dilution methods of Donovickand his co workers and of Price,
Nielsen, and Welch.
•Slide cell method of Heilmanfor estimating the amount of streptomycin in
body fluids.
•Turbidimetricmethod of Osgood and Graham.
•Streak plate method of Waksman and Reilly.

52. METHODS OF ASSAY
CHEMICAL METHODS by : --
•Schenck and Spielman.
•Scudi, Boxer and Jelinek.
•Levy, Schwed, and Sackett.
STRAINS USED: --
The test organism used depends upon the method and purpose of assay.
The special strains used are: --
1.Staphylococcus aureusand Bacillus subtilisin the agar diffusion assays.
2.Klebsiella pneumonia, Staphylococcus aureusand B. circulans in broth
dilution methods.
3.B. megatherium in the slide cell method.

53. INACTIVATION OF STREPTOMYCIN
•Chemical agents: --
1.Cysteine, B-mercaptoethylamine(sulfhydryl compounds) inhibited the
action of streptomycin against B.subtilis.
2.Hydrazine(0.002 M), phenylhydrazine(0.002 M), semicarbazide(0.002
M), methylphenylhydrazine(0.001 M), hydroxylamine(0.002 M)—ketone
reagents reduce the efficacy of streptomycin against either B.subtilis or
E.coli
3.Cevitamic acid(against A.aerogenes, S.aureus, E.coli, B.subtilis)
4.2 aminoethanediol

54. INACTIVATION OF STREPTOMYCIN
•Unfavorable reaction: --The use of a sugar, such as glucose, levulose, or
sucrose, medium which may be fermented by B.subtilis, E.coli, or other
organisms with the production of sufficient acid to reduce the pH, has
usually resulted in diminished activity on the part of streptomycin.
•Anaerobic conditions
•Age of culture: --Actively growing young cultures of bacteria are
apparently more susceptible to streptomycin than older ones(24 to 48
hrs. old) where the growth rate has decreased.

55. Compounds made from streptomycin
1.Streptomycin B: --this compound was isolated from streptomycin
concentrates by Fried and Titus. In the case of experimental infections
with M. tuberculosis, streptomycin B was about 1/3rd as active as
streptomycin on a weight basis and of about equal activity on a unit
basis.
2. Dihydrostreptomycin: --Peck, Hoffhine, Jr., and Folkers reported on
the formation of this compound by the catalytic hydrogenation of
streptomycin.

56. A PERFECT CURE FOR TUBERCULOSIS
For active tuberculosis it is often given together withisoniazid,rifampicin,
andpyrazinamide.It is not the first-line treatment, except in medically
under-served populations where the cost of more expensive treatments is
prohibitive. It may be useful in cases where resistance to other drugs is
identified.

57. OTHER USES OF STREPTOMYCIN
Treatment of diseases: --
•Infective endocarditiscaused by enterococcus when the organism is not
sensitive togentamicin.
•Plague(Yersinia pestis) has historically been treated with it as the first-
line treatment. However streptomycin is approved for this purpose only
by theU.S. Food and Drug Administration.

59. OTHER USES OF STREPTOMYCIN
•Inveterinary medicine, streptomycin is the first-line antibiotic for use
againstgram negativebacteria in large animals (horses,cattle,sheep,
etc.). It is commonly combined with procainepenicillinfor intramuscular
injection.
•Tularemiainfections have been treated mostly with streptomycin.
•Streptomycin is traditionally givenintramuscularly, and in many nations is
only licensed to be administered intramuscularly, though in some regions
the drug may also be administeredintravenously.

60. •Pesticide and fungicide
•Streptomycin also is used as a pesticide, to combat the growth of
bacteria, fungi, and algae. Streptomycin controls bacterial and fungal
diseases of certain fruit, vegetables, seed, and ornamental crops, and it
controlsalgaein ornamental ponds and aquaria. A major use is in the
control offire blighton apple and pear trees. As in medical applications,
extensive use can be associated with the development of resistant
strains.
•Cell culture
•Streptomycin, in combination with penicillin, is used in a standard
antibiotic cocktail to prevent bacterial infection in cell culture.
•Protein purification
•When purifying protein from a biological extract, streptomycin sulfate is
sometimes added as a means of removing nucleic acids. Since it binds to
ribosomes and precipitates out of solution, it serves as a method for
removing rRNA, mRNA, and even DNA if the extract is from a prokaryote.

61. BIOSYNTHESIS