ANTIBIOTICS (Tetracycline)

INTRODUCTION

The successful use of any therapeutic agent is compromised by the potential development of
tolerance or resistance to that compound from the time it is first employed. This is true for agents
used in the treatment of bacterial, fungal, parasitic, and viral infections and for treatment of chronic
diseases such as cancer and diabetes; it applies to ailments caused or suffered by any living
organisms, including humans, animals, fish, plants, insects, etc. A wide range of biochemical and
physiological mechanisms may be responsible for resistance. In the specific case of antimicrobial
agents, the complexity of the processes that contribute to emergence and dissemination of
resistance cannot be overemphasized, and the lack of basic knowledge on these topics is one of
the primary reasons that there has been so little significant achievement in the effective prevention
and control of resistance development. Most international, national, and local agencies recognize
this serious problem. Many resolutions and recommendations have been propounded, and
numerous reports have been written, but to no avail: the development of antibiotic resistance is
relentless. The most striking examples, and probably the most costly in terms of morbidity and
mortality, concern bacteria. The discovery of these infectious agents in the late 19th century
stimulated the search for appropriate preventative and therapeutic regimens; however, successful
treatment came only with the discovery and introduction of antibiotics half a century later.
Antibiotics have revolutionized medicine in many respects, and countless lives have been saved;
their discovery was a turning point in human history. Regrettably, the use of these wonder drugs
has been accompanied by the rapid appearance of resistant strains. Medical pundits are now
warning of a return to the pre-antibiotic era; a recent database lists the existence of more than
20,000 potential resistance genes (r genes) of nearly 400 different types, predicted in the main
from available bacterial genome sequences. Fortunately, the number existing as functional
resistance determinants in pathogens is much smaller. The antibiotic penicillin was discovered in
1928, but the complete structure of this relatively simple molecule was not revealed until 1949, by
the X-ray crystallographic studies of Dorothy Crowfoot Hodgkin, and was confirmed by total
synthesis in 1959. Studies of modes of action have provided biochemical information on ligands
and targets throughout antibiotic history, and the use of antibiotics as “phenotypic mutants” has
been a valuable approach in cell physiology studies.

The field of chemical biology/genetics grew from studies of those interactions. We have a
meager understanding of how antibiotics work, and in only a few instances can the intimate
interactions of the small molecule and its macromolecular receptor be interpreted in terms of
defined phenotypes. More surprisingly, there is a paucity of knowledge of the natural biological
functions of antibiotics, and the evolutionary and ecological aspects of their chemical and
biological reactions remain topics of considerable interest and value. To begin, the definition of
“antibiotic,” as first proposed by Selman Waksman, the discoverer of streptomycin and a pioneer
in screening of soils for the presence of biologicals, has been seriously overinterpreted; it is simply
a description of a use, a laboratory effect, or an activity of a chemical compound. It does not define
a class of compound or its natural function, only its application. At the risk of attack from purist
colleagues, the generic term “antibiotic” is used here to denote any class of organic molecule that
inhibits or kills microbes by specific interactions with bacterial targets, without any consideration
of the source of the particular compound or class. Thus, purely synthetic therapeutics are
considered antibiotics; after all, they interact with receptors and provoke specific cell responses
and biochemical mechanisms of cross-resistance in pathogens. The fluoroquinolones (FQs),
sulfonamides, and trimethoprim are good examples. As in any field of biological study, antibiotic
history is replete with misconceptions, misinterpretations, erroneous predictions, and other
mistakes that have occasionally led to the truth. This account aspires to focus on the truth.

Fig 2: classification and mechanism of action of antibiotic

The discovery of antibiotics is rightly considered one of the most significant health-related
events of modern times, and not only for its impact on the treatment of infectious diseases. Studies
with these compounds have often shown unexpected nonantibiotic effects that indicate a variety
of other biological activities; the result has been a significant number of additional therapeutic
applications of “antibiotics” as antiviral, antitumor, or anticancer agents. In some cases, the
alternative applications have surpassed those of antibiotic activity in importance, such as in the
treatment of cardiovascular disease or use as immunosuppressive agents. Unfortunately, the
colossal need for these valuable drugs has had a significant environmental downside. In the 60
years since their introduction, millions of metric tons of antibiotics have been produced and
employed for a wide variety of purposes. Improvements in production have provided increasingly
less expensive compounds that encourage nonprescription and off-label uses. The cost of the oldest
and most frequently used antibiotics is (probably) mainly in the packaging. The planet is saturated
with these toxic agents, which has of course contributed significantly to the selection of resistant
strains. The development of generations of antibiotic-resistant microbes and their distribution in
microbial populations throughout the biosphere are the results of many years of unremitting
selection pressure from human applications of antibiotics, via underuse, overuse, and misuse. This
is not a natural process, but a manmade situation superimposed on nature; there is perhaps no better
example of the Darwinian notions of selection and survival.

A LITTLE ANTIBIOTIC HISTORY

Since the introduction in 1937 of the first effective antimicrobials, namely, the sulfonamides,
the development of specific mechanisms of resistance has plagued their therapeutic use.
Sulfonamide resistance was originally reported in the late 1930s, and the same mechanisms operate
some 70 years later. A compilation of the commonly used antibiotics, their modes of action, and
resistance mechanisms is shown previously in Table 1.

Penicillin was discovered by Alexander Fleming in 1928, and in 1940, several years before the
introduction of penicillin as a therapeutic, a bacterial penicillinase was identified by two members
of the penicillin discovery team. Once the antibiotic was used widely, resistant strains capable of
inactivating the drug became prevalent, and synthetic studies were undertaken to modify penicillin
chemically to prevent cleavage by penicillinases (B-lactamases). Interestingly, the identification
of a bacterial penicillinase before the use of the antibiotic can now be appreciated in the light of
recent findings that a large number of antibiotic r genes are components of natural microbial
populations. Which came first, the antibiotic or resistance?
FIG. 2. History of antibiotic discovery and concomitant development of antibiotic resistance. The dark ages, the
preantibiotic era; primordial, the advent of chemotherapy, via the sulfonamides; golden, the halcyon years when most of
the antibiotics used today were discovered; the lean years, the low point of new antibiotic discovery and development;
pharmacologic, attempts were made to understand and improve the use of antibiotics by dosing, administration, etc.;
biochemical, knowledge of the biochemical actions of antibiotics and resistance mechanisms led to chemical modification
studies to avoid resistance; target, mode-of-action and genetic studies led to efforts to design new compounds; genomic/HTS,
genome sequencing methodology was used to predict essential targets for incorporation into high-throughput screening
assays; disenchantment, with the failure of the enormous investment in genome-based methods, many companies
discontinued their discovery programs. Other milestones in this history include the creation of the FDA Office of New Drugs
after the thalidomide disaster led to stricter requirements for drug safety, including the use of antibiotics. This slowed the
registration of novel compounds. Before antibiotics were discovered, Semmelweis advocated hand washing as a way of
avoiding infection; this practice is now strongly recommended as a method to prevent transmission.

In the case of streptomycin, introduced in 1944 for the treatment of tuberculosis (TB; “The
Great White Plague”), mutant strains of Mycobacterium tuberculosis resistant to therapeutic
concentrations of the antibiotic were found to arise during patient treatment. As other antibiotics
have been discovered and introduced into clinical practice, a similar course of events has ensued.
Figure 1 shows the sequence of discovery and resistance development for the major classes of
antibiotics. The unexpected identification of genetically transferable antibiotic resistance in Japan
in the mid-1950s (initially greeted with skepticism in the West) changed the whole picture by
introducing the heretical genetic concept that collections of antibiotic r genes could be
disseminated by bacterial conjugation throughout an entire population of bacterial pathogens (with
a few notable exceptions). Only in the past few years has it been appreciated that gene exchange
is a universal property of bacteria that has occurred throughout eons of microbial evolution. The
discovery of the presence of putative bacterial gene sequences in eukaryotic genomes has
heightened awareness of the great importance of horizontal gene transfer (HGT) in genome
evolution. Subsequently, other aspects of gene transfer have been revealed by the identification
and distribution of genomic islands carrying genes for pathogenicity, and other functional gene
clusters in different bacterial genera. Not surprisingly, plasmid-mediated transfer of antibiotic
resistance has been a major focus of investigation because of its medical and, more recently,
practical significance.

Production of Antibiotic

Antibiotic, which remains an important part of our antimicrobial armamentarium, had a

significant impact on the second half of the twentieth century. Deep-fermentation methods, which
were primarily developed for the production of penicillin during the war, gave rise to the
development of antibiotics and contributed to the nascent biotechnology industry which appeared
in the 1970s. Akin to other antimicrobials, penicillin is a secondary metabolite, thus it is only
produced in the stationary phase. The industrial production of penicillin was generally classified
into two processes – upstream processing and downstream processing. Upstream processing
encompasses any technology that leads to the synthesis of a product and includes the exploration,
development and production. The extraction and purification of a biotechnological product from
fermentation is referred to as downstream processing.

The Fermentation Process

Fermentation is the technique used for the commercial production of penicillin. It is a fed-batch
process that is carried out aseptically in stainless steel tank reactors with a capacity of 30 to 100
thousand gallons. The fermentation involves two to three initial seed growth phases, followed by
a fermentation production phase with a time cycle ranging from 120 to 200 hours. Various carbon
sources have been adopted for this process – including glucose, sucrose and other crude sugars.
Approximately 65% of the carbon is used for cellular maintenance, 25% for growth and only 10%
for penicillin production. Sugar is also used for the regulation of the pH value during active
penicillin production phase. Mini-harvest protocols are usually employed in penicillin
fermentation. They involve the removal of 20-40% of the fermentor contents and its replacement
with fresh sterile medium. This procedure can be repeated several times during this process without
yield reduction; quite the opposite, it can enhance the total penicillin yield per fermentor.
Antibiotic is excreted into the medium and recovered at the end of fermentation. Whole broth
extraction is best performed at acidic pH, with a 2-5% improvement in overall extraction
efficiency. Solvent extraction of chilled acidified broth is carried out with amyl, butyl or isobutyl
acetate. Present-day penicillin fermentations are highly automated and computerized. All the
necessary precursors, ammonia, sugar, carbon dioxide, oxygen are controlled, with thorough
monitoring of temperature and pH for optimal antibiotic production. The pH should be between
6.4 and 6.8 during the active production phase.

Pursuit for a Better Yield

When penicillin was initially made at the end of the Second World War using the fungus
Penicillium notatum, the process yielded one milligram per cubic decimeter. Today, with a use of
a different species (Penicillium chrysogenum) and improved extraction procedures the yield is 50
grams per cubic decimeter. The yield of penicillin can be further increased by improving the
composition of the medium, isolating aforementioned Penicillium chrysogenum which grows
better in huge deep fermentation tank, but also via the development of submerged culture technique
for cultivation of mold in large volume of liquid medium through which sterile air is forced. Still,
classical strain improvement has been the mainstay of penicillin production. The amplification of
the penicillin biosynthetic gene cluster between tandem repeats represents one of the most
important phenomena in high-yielding Penicillium chrysogenum strains. Molecular strategies that
differ from those involving an increase in biosynthesis gene doses have also been developed.

Modern Production Methods

Significant improvements in modern production methods have increased production and

decreased cost. Today, commercial producing strains of Penicillium chrysogenum are grown using
submerged culture in constantly agitating and aerated 50,000-gallon stainless steel tanks. These
industrial strains can now produce 40-50 grams of penicillin per liter of culture with a 90%
recovery yield.
 Fig3: commercial production of antibiotic

This is an overwhelming improvement from the earliest Peoria farmer’s market strain that only
produced 0.15 grams per liter with very low recovery rates. In order to achieve these production
rates, modern Penicillium strains display a host of genetic and cellular modifications that result in
increased production, including amplification of the penicillin biosynthesis gene cluster, an
increased number of peroxisomes, and elevated levels of transporter proteins that secrete newly
produced penicillin out of the peroxisomes and the cell. Worldwide sales of penicillin and other
beta-lactam antibiotics is now greater than $15 billion (U.S. dollars) per year. These sales numbers
exist despite the fact that cost is now at an all-time low. Penicillin now costs $10 per kilogram
versus $300 per kilogram in 1953. Although Europe is the major producer of beta-lactam
antibiotics, newer manufacturing facilities are relocating to China and other regions of Asia where
labor and energy costs are lower.
Tetracyclines
Tetracycline’s: A family of broad-spectrum antibiotics effective against a remarkably wide
variety of organisms. Bacteria susceptible to tetracycline include H. flu (Haemophilus influenzae),
strep (Streptococcus pneumoniae), Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia
trachomatis, and Neisseria gonorrhoeae (the cause of gonorrhea). Tetracycline is also used to treat
nongonococcal urethritis (due to Ureaplasma), Rocky mountain spotted fever, typhus, chancroid,
cholera, brucellosis, anthrax, and syphilis. It is used in combination with other medications to treat
Helicobacter pylori, the bacteria associated with ulcers of the stomach and duodenum.

Fig 4: chemical structure of tetracycline family

Historical background:-

The first member of the group to be discovered is chlortetracycline (Aureomycin) in the late
1940s by Benjamin Minge Duggar, a scientist employed by American Cyanamid - Lederle
Laboratories, under the leadership of Yellapragada Subbarow, who derived the substance from a
golden-colored, fungus-like, soil-dwelling bacterium named Streptomyces aureofaciens.
Oxytetracycline (Terramycin) was discovered shortly afterwards by AC Finlay et al.; it came from
a similar soil bacterium named Streptomyces rimosus. Robert Burns Woodward determined the
structure of oxytetracycline, enabling Lloyd H. Conover to successfully produce tetracycline itself
as a synthetic product. The development of many chemically altered antibiotics formed this group.
In June 2005, tigecycline, the first member of a new subgroup of tetracyclines named
glycylcyclines, was introduced to treat infections that are resistant to other antimicrobics including
conventional tetracyclines. While tigecycline is the first tetracycline approved in over 20 years,
other, newer versions of tetracyclines are currently in human clinical trials. A research conducted
by anthropologist George J. Armelagos and his team at Emory University showed that ancient
Nubians from the post-Meroitic period (around 350 CE) had deposits of tetracycline in their bones,
detectable through analyses of cross sections through ultraviolet light - the deposits are fluorescent,
just as modern ones. Armelagos suggested that this was due to ingestion of the local ancient beer
(very much like the Egyptian beer), made from contaminated stored grains.

Industrial production of Tetracycline

The production of preponderant amounts of tetracycline by the addition of a bromide or the like,
e. g. sodium bromide, to such fermentations has been disclosed by Lein and Gourevitch in Belgian
Patent 533,886. The production by fermentation of relatively large amounts of tetracycline as
compared to the amount of chlortetracycline has been achieved by Minieri et a1. by the use of
dechlorinated corn steep liquor in a medium which otherwise contains practically no chloride ions,
as disclosed in South African application and Belgian Patent. In the process of Minieri, the most
potent broths were obtained only by the use of corn steep liquor which had been dechlorinated in
laborious and expensive fashion and in a manner which still gave broths of relatively low potency.
The production of tetracycline by fermentation of Streptomyces wureofaciens or Streptomyces
viridifaciens which provide increased total amounts or" tetracycline and also provide a very high
ratio of tetracycline to chlortetracycline, i. e. equal to or greater than nine to one, Without the
disadvantages in expense, additional labor, increased cycle time, added chemicals, lowered
potency and increased ditficulty of purification introduced by the use of dechlorinating agents or
inhibitors such as bromide ion. The production of tetracycline which comprises aerobically
growing a culture of a chlortetracycline-producing species of Streptomyces selected from the
group consisting of Streptomyces aureofaciens and Srreptomyces viridifaciens in an aqueous
carbohydrate solution containing as organic sources of nitrogen from 0.1 to 5.0 percent by weight
of soybean meal, from 0.1 to 5.0 percent by weight of substantially oil-free cottonseed endosperm
flour, from 0.1 to 5.0 percent by weight of corn oil meal Patented Jan. 1, 1957 and from 0.1 to 2.0
percent by weight of yeast, said fermentation medium being un-dechlorinated, and said
tetracycline in the final fermentation broth comprising greater than percent of the antibiotic activity
and being present in the amount of at least 2500 meg/ml. Tetracycline is thus prepared by
cultivation under said particular, controlled conditions of the chlortetracycline producing species
of Streptomyces, i. e. Streptomyces aureofaciens (NRRL 2209) and Streptomyces viridifaciens
(ATCC 11989); the latter is described in U. S. Patent 2,712,517. Especially included in the present
invention is the use of various strains and natural isolates of these species of Streptomyces as Well
as mutants produced therefrom by mutating agents such as X-radiation, ultra-violet radiation,
nitrogen mustards and similar chemicals, etc.

Other than with respect to the unique combination of organic nitrogen sources of the present
invention, the fermentation is carried out using the medium constituents and processes known to
be useful in the preparation by submerged aerobic fermentation of other broad spectrum antibiotics
such as chlortetracycline and oxytetracyclinc. Thus, use is made of sugars and glyceride oils as
carbon sources, of buffering agents such as calcium carbonate, of inorganic sources of nitrogen
such as ammonium sulfate, and of trace amounts of metallic ions such as zinc; no compounds
containing chloride ion are used, of course. All of these media constituents, other than the organic
nitrogen sources, are virtually chloride-free and available commercially.

In the practice of the present invention, use is made in the medium of from 0.1 to 5.0 percent by
Weight of soybean meal, and preferably of 0.5 to 3.0 percent. The amount of yeast to be used is
about 0.1 to 2.0 percent and preferably about 0.1 to 0.4 percent; any yeast such as Sacclzaromyces
cerivisiae or Torula spp. may be used and specific examples include debittered brewers yeast and
primary grown bakers yeast. The corn oil meal is used in the amount of 0.1 to 5.0 percent, and
preferably from 0.25 to 2.0 percent. Substantially oil-free cottonseed endosperm flour is used in
the amount of 0.1 to 5.0 percent, and preferably about 1.0 to 2.0 percent. The cottonseed endosperm
flour is available commercially (e. g. as Pharmamedia or Proflo from Traders Oil Mill Co., Fort
Worth, Texas) and is prepared by cooking dehulled cottonseeds, extruding at least percent of the
oil therein, and collecting by air-settling the endosperm, which is then ground to a flour, i. e. 370
mesh. Thus the phrase substantially oil-free refers to the fact that this flour contains less than five
percent of the original content of oil of the cottonseed. This cottonseed endosperm ilour is an
entirely different material than cottonseed meal, which is not as suitable for use in the production
of tetracycline because it leads to production of too much chlortetracycline.

The phrase un-dechlorinated medium as used herein refers to a complete medium, or any
constituent or mixture of constituents thereof, which have not been subjected to any chemical or
physical process which removes chloride ion effectively, either before or after preparation or" the
final medium. The avoidance of such dechlorinating procedures is one of the most important
advantages of the process of the present invention. This process would be of no practical use,
however, if the. portant advantage of the process of the present invention 3 that the ratio of
tetracycline to chlortetracycline produced thereby is greater than nine to one. Chloride-free media
of the synthetic type, i. e. those which use individual, isolated amino-acids or peptides as a source
of organic nitrogen, cannot produce broths containing as much as 500 meg/ml. and are thus of no
commercial interest. It is therefore a further advantage of the process of the present invention that
it produces final fermentation broths containing at least 2500 meg/ml. tetracycline. It is preferred,
but not essential, that the inoculum used for the fermentation of the present invention also be grown
in an undechlorinated medium of the type used in the final fermentation. The following examples
are provided for purposes of illustration only and are not to be construed as limiting the invention.

- Example I Streptomyces aureofacicns was fermented for 100 hours at 85 F., using 74 cubic
feet per minute of air, no agitation, lard oil defoamer containing 2% octadccanol and 7.5%
mineral oil and 60 gallons of vegetative inoculum, in about 600 gallons of a medium, sterilized
25 minutes at 250 F., containing 6% sucrose, 2% soybean meal, 1.5% cottonseed endosperm
flour (Pharmamedia), corn oil meal or dechlorinated corn steep liquor in amounts indicated
below, 0.2% brewers debittered yeast, 0.5% ammonium sulfate, and 1.0% calcium carbonate
with the following tabulated results: Additional Medium Constituents Antibiotic Ratio of
Activity Tetracycline in meg/ml. t chlortetra- Run Number Dechlori- Tetracycline cycline by
uated Corn Oil Hydrochloride Paper Strip Corn Steep Meal, Equivalents Chromatog- Liquor,
Percent raphy Percent The media in runs 15 inclusive also contained 0.003% zinc sulfate
heptahydrate and 0.06% of an ironsequestering agent (versene, Type Fe-3 specific). The
inoculum in run 14 was prepared in a medium containing no dechlorinated corn steep liquor.
- Example II & III Use of 2.0% cottonseed endosperm flour, 1.0% calcium carbonate and
1.0% soybean meal gave over 2500 meg/ml. of tetracycline equivalents and a tetracycline to
chlortetracycline ratio of greater than 90:10 when used with 0.5% or 1.0% corn oil meal in
shake flask fermentations containing no corn steep liquor and conducted otherwise according
to Example I but on a smaller scale and with the aeration supplied by shaking. The other
constituents of the medium were 6% sucrose, 0.2% debittered brewers yeast, 0.5% ammonium
sulfate, 0.003% ZnSO4-7Hz0 and 0.06% of iron chelating agent (Fe+++ specific Versene).
- Example IV Use of 1.0% cottonseed endosperm flour, no calcium carbonate and 1.0% or
2.0% soybean meal gave from 2805-3100 meg/ml. tetracycline equivalents and a tetracycline
to chlortetracycline ratio of greater than :10 when used with 0.25%, 0.50% or 1.0% corn oil
meal in shake flask fermentations containing no corn steep liquor and conducted otherwise
according to Example I but on a smaller scale and with the aeration supplied by shaklng. The
other constituents of the medium were 6% sucrose, 0.2% debittered brewers yeast, 0.5%
ammonium sulfate, 0.003% ZnSO4-7H2O and 0.06 of ironchelating agent (Fe+++ specific
Versene).
 Extensive experimental work has demonstrated that omission of any one or more of the four
organic sources of nitrogen, i. e., cottonseed endosperm flour, soybean meal, corn oil meal or yeast,
from these un-dechlorinated media, or substitution of another agent therefor, resulted in either a
large reduction in total potency, i. e., well below 2500 mcg./ml., or a decrease below nine to one
of the ratio of tetracycline to chlortetracycline. Mutants and strains of Streptomyces viridz'faciens
which produce 2500 meg/ml. chlortetracycline in the media of Niedercorn and Petty can be used
with equal efifectiveness in place of the Streptomyces aureofaciens in the examples above.

There is a many other proposed processes for production of tetracycline which include:-

1) A process for the production of tetracycline which comprises aerobically growing a culture of
a chlortetracycline-producing species of Streptomyces selected from the group consisting of
Streptomyces aureofaciens and Streptomyces vilidifaciens in an aqueous carbohydrate solution
containing as organic sources of nitrogen from 0.1 to 5.0 % by weight of soybean meal, from
0.1 to 5.0 percent by weight of substantially oil-free cottonseed endosperm flour, from 0.1 to
5.0 percent by weight of corn oil meal and from 0.1 to 2.0 % by weight of yeast, said
fermentation medium being undechlorinated and said tetracycline in the final fermentation
broth comprising greater than 90 % of the antibiotic activity and being present in the amount
of at least 2500 mcg./ ml.
2) A process for the production of tetracycline which comprises aerobically growing a culture of
a chlortetracycline-producing species of Streptomyces selected from the group consisting of
Streptomyces aureofacicns and Streptomyces viridifaciens in an aqueous carbohydrate
solution containing as organic sources of nitrogen from 0.5 to 3.0 % by Weight of soybean
meal, from 1.0 to 2.0 % by weight of substantially oil-free cottonseed endosperm flour, from
0.25 to 2.0 % by weight of corn oil meal and from 0.1 to 0.4 % by weight of yeast, said
fermentation medium being un-dechlorinated and said tetracycline in the final fermentation
broth comprising greater than 90 % of the antibiotic activity and being present in the amount
of at least 2500 meg/ml.
3) A process for the production of tetracycline which comprises aerobically growing a culture of
Streptomyces aureofaciens in an aqueous carbohydrate solution containing as organic sources
of nitrogen from 0.1 to 5.0 % by weight of soybean meal, from 0.1 to 5.0 % by weight of
substantially oil-free cottonseed endosperm flour, from 0.1 to 5.0 % by weight of corn oil meal
 and from 0.1 to 2.0 % by weight of yeast, said fermentation medium being un-dechlorinated
and said tetracycline in the final fermentation broth comprising greater than 90 % of the
antibiotic activity and being present in the amount of at lea-st 2500 mcg./ ml.
4) A process for the production of tetracycline which comprises aerobically growing a culture of
Streptomyces aureofaciens in an aqueous carbohydrate solution containing as organic sources
of nitrogen from 0.5 to 3.0 % by weight of soy bean meal, from 1.0 to 2.0 % by weight of
substantially oil-free cottonseed endosperm flour, from 0.25 to 2.0 % by weight of corn oil
meal and from 0.1 to 0.4 % by weight of yeast, said fermentation medium being un-
dechlorinated and said tetracycline in the final fermentation broth comprising greater than 90
% of the antibiotic activity and being present in the amount of at least 2500 meg/ml.
5) A process for the production of tetracycline which comprises aerobically growing a culture of
Streptomyces viridifaciens in an aqueous carbohydrate solution containing as organic sources
of nitrogen from 0.1 to 5.0 % by weight of soybean meal, from 0.1 to 5.0 % by weight of
substantially oil-free cottonseed endosperm flour, from 0.1 to 5.0 % by weight of corn oil meal
and from 0.1 to 2.0 % by weight of yeast, said fermentation medium being un-dechlorinated
and said tetracycline in the final fermentation broth comprising greater than 90 % of the
antibiotic activity and being present in the amount of at least 2500 mcg / ml.
6) A process for the production of tetracycline which comprises aerobically growing a. culture of
Streptomyces viridifaciens in an aqueous carbohydrate solution containing as organic sources
of nitrogen from 0.5 to 3.0 % by weight of soy bean meal, from 1.0 to 2.0 % by weight of
substantially oil-free cottonseed endosperm flour, from 0.25 to 2.0 % by Weight of corn oil
meal and from 0.1 to 0.4 % by Weight of yeast, said fermentation medium being un-
dechlorinated and said tetracycline in the final fermentation broth comprising greater than 90
% of the antibiotic activity and being present in the amount of at least 2500 meg/ml.

Medical uses:-

Tetracyclines are generally used in the treatment of infections of the urinary tract, respiratory
tract, and the intestines and are also used in the treatment of chlamydia, especially in patients
allergic to β-lactams and macrolides; however, their use for these indications is less popular than
it once was due to widespread development of resistance in the causative organisms. Their most
common current use is in the treatment of moderately severe acne and rosacea (tetracycline,
oxytetracycline, doxycycline or minocycline). Doxycycline is also used as a prophylactic treatment
for infection by Bacillus anthracis (anthrax) and is effective against Yersinia pestis, the infectious
agent of bubonic plague. It is also used for malaria treatment and prophylaxis, as well as treating
elephantitis filariasis. Tetracyclines remain the treatment of choice for infections caused by
chlamydia (trachoma, psittacosis, salpingitis, urethritis and L. venereum infection), Rickettsia
(typhus, Rocky Mountain spotted fever), brucellosis and spirochetal infections (borreliosis,
syphilis and Lyme disease). In addition, they may be used to treat anthrax, plague, tularemia and
Legionnaires' disease. They are also used in veterinary medicine. They may have a role in reducing
the duration and severity of cholera, although drug-resistance is mounting, and their effect on
overall mortality is questioned. Tetracycline derivatives are currently being investigated for the
treatment of certain inflammatory disorders.

Mechanism of Action of Tetracyclines

Tetracyclines act by binding to the 30S subunit of the ribosome at the A-site. During protein
biosynthesis, the new t-RNA with the amino acid attempts to bind to A-site of the ribosome.
However, since the A-site is blocked by the tetracycline, the aminoacyl-tRNA cannot bind to it.
Thus without the sequential attachment of the tRNA at the A-site, protein biosynthesis cannot
occur. By inhibiting protein biosynthesis tetracyclines cause cell death of the bacterial cell.

Fig 5: Mechanism of Action of Tetracyclines

 The above animations has been supplied by Dr. Gary E. Kaiser from the Community College of
Baltimore County– and it illustrates the mechanism of action of tetracyclines. The transcript of the
animation is as follows: ” The tetracyclines (tetracycline, doxycycline, demeclocycline,
minocycline, etc.) block bacterial translation by binding reversibly to the 30S subunit and
distorting it in such a way that the anticodons of the charged tRNAs cannot align properly with the
codons of the mRNA.

Mechanism of resistance

Tetracycline inhibits cell growth by inhibiting translation. It binds to the 16S part of the 30S
ribosomal subunit and prevents the amino-acyl tRNA from binding to the A site of the ribosome.
The binding is reversible in nature. Cells become resistant to tetracycline by at least three
mechanisms: enzymatic inactivation of tetracycline, efflux, and ribosomal protection. Inactivation
is the rarest type of resistance, where an acetyl group is added to the molecule, causing inactivation
of the drug. In efflux, a resistance gene encodes a membrane protein that actively pumps
tetracycline out of the cell. This is the mechanism of action of the tetracycline resistance gene on
the artificial plasmid pBR322. In ribosomal protection, a resistance gene encodes a protein that
can have several effects, depending on what gene is transferred. Six classes of ribosomal protection
genes/proteins have been found, all with high sequence homology, suggesting a common
evolutionary ancestor.

Possible mechanisms of action of these protective proteins include:

- blocking tetracyclines from binding to the ribosome

- binding to the ribosome and distorting the structure to still allow t-RNA binding while
tetracycline is bound
- binding to the ribosome and dislodging tetracycline.

All of these changes to ribosomes are reversible (non-covalent) because ribosomes isolated from
both tetracycline-resistant and susceptible organisms bind tetracycline equally well in vitro.

Pharmacokinetics
Tetracyclines mainly differ in their absorption after oral administration and their elimination.
Absorption after oral administration is approximately 60–70% for tetracycline, oxytetracycline,
and methacycline; and 95–100% for doxycycline and minocycline. A portion of an orally
administered dose of tetracycline remains in the gut lumen, modifies intestinal flora, and is
excreted in the feces. Absorption occurs mainly in the upper small intestine and is impaired by
food (except doxycycline and minocycline); by divalent cations (Ca2+, Mg2+, Fe2+) or Al3+; by
dairy products and antacids, which contain multivalent cations; and by alkaline pH.

Tetracyclines are 40–80% bound by serum proteins. Tetracyclines are distributed widely to tissues
and body fluids except for CSF, where concentrations are 10–25% of those in serum. Minocycline
reaches very high concentrations in tears and saliva, which makes it useful for eradication of the
meningococcal carrier state. Tetracyclines cross the placenta to reach the fetus and are also
excreted in milk. As a result of chelation with calcium, tetracyclines are bound to and damage –
growing bones and teeth. Carbamazepine, phenytoin, barbiturates, and chronic alcohol ingestion
may shorten the half-life of doxycycline by 50% by induction of hepatic enzymes that metabolize
the drug. It excreted mainly in bile and urine. Concentrations in bile exceed those in serum tenfold.
Some of the drug excreted in bile is reabsorbed from the intestine (enterohepatic circulation) and
may contribute to maintenance of serum levels. From 10 to 50% of various tetracyclines is
excreted into the urine, mainly by glomerular filtration. Ten to 40% of the drug is excreted in
feces. Doxycycline, in contrast to other tetracyclines, is eliminated by nonrenal mechanisms, do
not accumulate significantly and require no dosage adjustment in renal failure. Tetracyclines and
macrolides have a good intracellular distribution.

Side effects of Tetracycline's

- Severe headache, dizziness, blurred vision.

- Fever, chills, body aches, flu symptoms.
- Severe blistering, peeling, and red skin rash.
- Urinating less than usual or not at all.
- Pale or yellowed skin, dark colored urine, fever, confusion or weakness.
- Severe pain in your upper stomach spreading to your back, nausea and vomiting, fast heart
rate.
- Loss of appetite, jaundice (yellowing of the skin or eyes).
- Easy bruising or bleeding, unusual weakness.
Adverse Reactions

Nausea, anorexia, and diarrhea can usually be controlled by administering the drug with food or
carboxymethylcellulose, reducing drug dosage, or discontinuing the drug. Tetracyclines modify
the normal flora, with suppression of susceptible coliform organisms and overgrowth of
pseudomonas, proteus, staphylococci, resistant coliforms, clostridia, and candida. This can result
in intestinal functional disturbances, anal pruritus, vaginal or oral candidiasis, or enterocolitis with
shock and death. Tetracyclines are readily bound to calcium deposited in newly formed bone or
teeth in young children. When a tetracycline is given during pregnancy, it can be deposited in the
fetal teeth, leading to fluorescence, discoloration, and enamel dysplasia; it can be deposited in
bone, where it may cause deformity or growth inhibition. If the drug is given for long periods to
children under 8 years of age, similar changes can result. Tetracyclines can probably impair hepatic
function, especially during pregnancy, in patients with preexisting hepatic insufficiency and when
high doses are given intravenously. Hepatic necrosis has been reported with daily doses of ≥ 4 g
i.v. Renal tubular acidosis and other renal injury resulting in nitrogen retention is a conraindication
to the administration of outdated tetracycline preparations. Tetracyclines given with diuretics may
produce nitrogen retention. Tetracyclines other than doxycycline may accumulate to toxic levels
in patients with impaired kidney function. Intravenous injection can lead to venous thrombosis.
Intramuscular injection produces painful local irritation and should be avoided. Systemically
administered tetracycline, especially demeclocycline, can induce sensitivity to sunlight or
ultraviolet light, particularly in fair-skinned persons. Dizziness, vertigo, nausea, and vomiting have
been noted particularly with doxycycline and Minocycline at high doses.
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