Penicillin is one of the most commonly used antibiotics globally, as it has a wide range of clinical indications. Penicillin is effective against many different types of infections involving gram-positive cocci, gram-positive rods (e.g., Listeria), most anaerobes, and gram-negative cocci (e.g., Neisseria). Importantly, certain bacterial species have obtained penicillin resistance, including enterococci. Enterococci infections now receive treatment with a combination of penicillin and streptomycin or gentamicin. Certain gram-negative rods are also resistant to penicillin due to penicillin’s poor ability to penetrate the porin channel. However, later generations of broad-spectrum penicillins are effective against gram-negative rods. Second-generation penicillins (ampicillin and amoxicillin) can also penetrate the porin channel, making these drugs effective against Proteus mirabilis, Shigella, H. influenzae, Salmonella, and E. coli. Third-generation penicillins such as carbenicillin and ticarcillin are also able to penetrate gram-negative bacterial porin channels. Fourth-generation penicillins such as piperacillin are effective against the same bacterial strains as third-generation penicillins as well as Klebsiella, enterococci, Pseudomonas aeruginosa, and Bacteroides fragilis.

2.  Introduction
 History
 Structure
 Mode of action
 Fermentation and production of penicillin
 Administration
 Adverse effects
 Contraindication
 Penicillin resistance
 Advantages and disadvantages
 Toxicity
 Conclusion
 References

3.  ‘Antibiotic’ means against life – antibiotics only kill life that
is harmful to living creatures
 Penicillin is a group of antibiotics that are commonly used to
treat different types of gram positive bacterial infections
 Also called beta-lactum antibiotics
 Penicillin is a secondary metabolite derived from the
penicillin mould
 It destroys bacteria by inhibiting the enzymes responsible for
the formation of cell wall in bacterial cells

4. Penicillin

5. Scientific classification
Kingdom: Fungi
Division: Ascomycota
Class: Eurotiomycetes
Order: Eurotiales
Family: Trichocomaceae
Genus: Penicillium
Species: chrysogenum
Binomial name
Penicillium chrysogenum Penicillium chrysogenum in PDA

6.  1928-ALEXANDER FLEMING
bread mould (penicillium
notatum) growing on petri dish
 1939-FLOREY ,CHAIN and
associates began to work on
isolating and synthesizing large
amounts of penicillin.
 1941-Penicillin is used clinically
as antibiotic
ALEXANDER FLEMING

7.  They have a basic ring like
structure called Beta-lactum
ring derived from two amino
acid valine and cysteine via
tripeptide intermediate .
 The third amino acid of this
tripeptide is replaced by
acyl group
 The nature of this acyl group
produces specific properties
on different types of
penicillin

10.  Most bacteria have a peptidoglycan cell wall that surrounds the bacterial
plasma membrane, prevents osmotic lysis, and provides structural
integrity. The peptidoglycan wall is continually remodeling during
replication and growth. Penicillin inhibits the cross-linking of
peptidoglycan in the cell wall.
 The catalyst for this reaction is penicillin-binding proteins, such as the
enzyme DD-transpeptidase. Penicillin's four-membered β-lactam ring
can bind to DD-transpeptidase to irreversibly inactive it. The bacteria,
therefore, are unable to build their cell walls even while other proteins
continue to break down the wall.

11.  As the bacteria cell wall continues to weaken, osmotic pressure pushes
water into the cell and kills the cell. Peptidoglycan fragments further
destroy the cell wall as these fragments can activate autolysins and
hydrolases.
 The penicillins can also be combined with a beta-lactamase inhibitor
such as clavulanic acid to enhance its effects. Beta-lactamase inhibitors
prevent the degradation of the beta-lactam ring in penicillin that can
occur when certain species of bacteria express the enzyme beta-
lactamase.

13. Fermentation process can be described under four steps:
1. Strain development
2. Inoculum production
3. Inoculation
4. Extraction and purification

15. 1)Strain development
 It is highly desirable to use high yielding strain in manufacture of
antibiotic. This can be achieved by Sequential genetic selection
 In other words such a strain can be obtained by step-wise
development with the help of a series of mutagenic treatments or
exposing to UV radiation is called strain improvement
 Each followed by the selection of improved mutants
 This mutants possesses a far greater capacity for antibiotic
production than the wild strain

16.  It has been found that high yielding strains of penicillium chrysogenum
are genetically unstable therefore, they are carefully maintained.
 Production strains are stored in dormant form by
 Spore suspension can be lyophilised in appropriate media
 Spore suspensions can be stored under liquid nitrogen in frozen state

17. Improved strain yield
P. chrysogenum, NRRL
1951.B25
200 units/ml
P. Chrysogenum X-1612-
B25 x ray irradiation
500 units/ml
P. chrysogenum, Q-176-
treatment of conidia with UV
light
761 units/ml

18. 2)Inoculum Production:
 The microorganism which is used in a fermentation process is
called as the inoculum.
 A high yielding strain of P. chrysogenum is generally employed
as inoculum.
 A strain of the fungus is sub-cultured from stock culture for
inoculum development. Spores from primary source are
suspended in a dilute solution of a nontoxic wetting agent such
as 1:10000 sodium lauryl sulfate + water.
 The spores are then added to plates of sporulation medium and
these are incubated for five to seven days at 24°C so as to
provide heavy sporulation. The entire process is repeated several
times in order to have more sporulation.

19. Sporulation medium- Moyer and Coghill(1946)
Components g/L
Glycerol 7.5
Cane molasses 7.5
Corn-steep liquor 2.5
MgSO4.7H2O 0.050
KH2PO4 0.060
Peptone 5.00
NaCl 4.00
Fe-Tartrate 0.005
CuSO4-5H20 0.004
Agar 2-50
Distilled water to make 1.0 ltr

20. 3. Innoculation
 By suspension of ungerminated mould spores into a non toxic
wetting agent for uniform spore suspension (sodium lauryl
sulfonate+ sterile water 1:10,000). Followed by aeration
,agitation –equal distribution of spore suspension
 Feeding the fermentation tanks with pre-germinated spores
which are prepared by germination of spores
 After inoculation into inoculum tanks or stirred fermenters, The
incubation temperature is maintained at 24-27°C for 2 days with
agitation and aeration in order to facilitate heavy mycelial
growth, which may be added to a second or even a third stage
fermentation.
 The resulting inoculum which is employed in a production tank
is tested both by microscopic examination and by sub-culturing
method.

21. The medium employed for penicillin production should be suitable to
achieve:
1. An abundant growth of the mycelium.
2. Maximum accumulation of the antibiotic.
3. Easy and inexpensive extraction and purification of the antibiotic.

22.  Carbon source
Lactose in a concentration of 6%
Other carbohydrates like glucose and galactose
 Nitrogen source
Ammonium salts such as ammonium sulphate, ammonium lactate ,
ammonia gas are used
 Mineral source
These elements include phosphorous ,sulphur , magnesium ,zinc, iron
and copper

23. PRECURSOR
 The most important naturally occurring penicillins are penicillin G
(benzyl-penicillin) and penicillin-V ( phenoxymethyl-penicillin)
 The formation of a desirable penicillin can be stimulated by addition of
phenylacetic acid derivatives .
 Eg: The mould, penicillium chrysogenum synthesizes large quantities of
penicillin G if phenyl acetic acid is present in the fermentation medium .
 PAA supplies side chain of penicillin-G

24.  Penicillin production is an aerobic process and therefore, a continuous
supply of O2 to the growing culture is very essential.
 The required aeration rate is 0.5-1.0 vvm.
 pH is maintained around 6.5, and the optimal temperature is in the range
of 25-27°C. Penicillin production is usually carried out by submerged
process.
 Phenyl acetic acid or pehnoxyacetic acid is fed continuously as
precursor.

25.  Phase I (trophophase):
 rapid growth occurs, lasts for about 30 hours during which mycelia
are produced.
 Phase II(idiophase) :
 lasts for five to seven days;
 growth is reduced and penicillin is produced.
 Phase III:
 carbon and nitrogen sources are depleted,
 antibiotic production ceases,
 the mycelia lyse releasing ammonia and the pH rises.

26.  After it is assessed that sufficient amount of penicillin has been
produced during fermentation process, it is extracted and then
purified.
 The entire process is carried out in three different stages.
 They are:
1) Separation of mycelium
2) Extraction of penicillin and
3) Treatment of crude extract

27. 1)Separation of Mycelium:
 Mycelium is separated from the medium by employing rotatory vaccum
filter. This process should be performed carefully in order to avoid
contaminating microorganisms which produce penicillinase enzyme,
degrading the penicillin.
2)Extraction of penicillin
 Extraction of penicillin is carried out by employing counter current
extraction method.
 The pH of the liquid after separation of the mycelium is adjusted to 2.0
to 2.5 by adding sulphuric acid. This treatment converts penicillin into
anionic form.
 The liquid is immediately extracted with an organic solvent such as
amylacetate or butylacetate

28.  This step has to be carried out quickly because penicillin is quite unstable
at low pH values.
 The penicillin is then back extracted into buffer by adding enough
potassium or sodium hydroxide which also results in the elevation of pH
to 7.0 to 7.5.
 The resulting aqueous solution is acidified and re-extracted with organic
solvent. These shifts between the water and the solvent and help in
purification of penicillin
 Finally the penicillin obtained is sodium penicillin

30. 3) Treatment of Crude Extract:
 The resulted sodium penicillin is treated with charcoal to remove
pyrogens (fever causing substances). It is also, sometimes, sterilized to
remove bacteria by using Seitz filter. Then, the sodium penicillin is
prepared in crystalline form by crystallization.
 The antibiotic is then packed in sterile vials as powder or suspension
 For oral use it is tableted with a film coating
 Tests (Eg. For potency, purity, freedom from the pyrogens and sterility )
are performed on finished product before being marketed

32.  Penicillin G administration can be either intravenously or
intramuscularly. Penicillin G benzathine administration ensures a
continuous low dose of penicillin G over 2 to 4 weeks. Penicillin V and
penicillin VK (potassium salt of penicillin V) is available in an orally
administered form. As with any antibiotic, patients must receive counsel
to finish the full course of medicine to prevent bacterial resistance.
 Oral vs. injection will have different bio availabilities. Penicillin G
degrades more easily by stomach acid and has a bioavailability of less
than 30%. Therefore, it is a parenterally administered drug. Penicillin V
has a bioavailability of around 65% after passing stomach acid.
Penicillin V is best administered to a fasting patient as it degrades in
stomach acid.
 Penicillin demonstrates limited crossing of the blood-brain barrier and
can only treat some bacterial meningitis. Most penicillin derivatives are
not metabolized much by the liver. They are rapidly excreted in the urine
as they are water-soluble, and some of the drug is excreted in bile.
Penicillin has a relatively short half-life of about 2 hours.

33.  Penicillin V and G both can have adverse effects, including
nausea, vomiting, diarrhea, rash, abdominal pain, and urticaria.
Penicillin G can have additional effects of muscle spasms, fever,
chills, muscle pain, headache, tachycardia, flushing, tachypnea,
and hypotension.
 GI symptoms were the most common and were reported in over
1% of patients, while hypotension, urticaria, and anaphylaxis are
severe but rare side effects. Symptoms of rash can appear a week
after initiating therapy.
 The penicillins can also cause acute interstitial nephritis, a
disease characterized by inflammation of the tubules and
interstitium of the kidneys. Acute interstitial nephritis can also
present with hematuria, fever, and rash. In this situation, the
recommendation is to withdraw the drug as the disease could
lead to renal failure

34.  Contraindications of penicillin include a previous history of
severe allergic reaction or penicillin and its derivatives. Penicillin
is also contraindicated in patients who have had Stevens-Johnson
syndrome after administering penicillin or a penicillin derivative.
 The penicillins are safe to use during pregnancy and nursing, as
the drug appears at a low concentration in breast milk. Although
renal impairment is not a contraindication for penicillin, doses
will have to be adjusted given end-stage renal disease. These
patients will receive a full loading dose and then half a loading
dose every 8 to 10 hours or 4 to 5 hours, depending on the
glomerular filtration rate.
 Penicillin has an antagonistic effect with tetracycline and
reportedly can lead to a 2.6 times greater risk for mortality when
treating pneumococcal meningitis compared to using penicillin
alone. Penicillin requires bacterial cell wall synthesis to be active
to be effective.

35.  The first sign of antibiotic resistance became apparent soon after the
discovery of penicillin. In 1940, Abraham and Chain reported that an E.
coli strain was able to inactivate penicillin by producing penicillinase.
 The spread of penicillin resistance was already documented by 1942,
when four Staphylococcus aureus strains were found to resist the action
of penicillin in hospitalized patients.
 During the next few years, the proportion of infections caused by
penicillin-resistant S. aureus rapidly rose, spreading quickly from
hospitals to communities. By the late 1960s, more than 80 percent of
both community and hospital-acquired strains of S. aureus were
penicillin-resistant.

36.  The rapid spread of penicillin resistance temporarily came to a
halt after the introduction of the second-generation, semisynthetic
methicillin in the 1960s. However, methicillin-resistant strains
soon emerged, and only in 1981 was this mechanism of resistance
unraveled: these strains harbored an altered PBP, designated PBP-
2a, which showed a reduced affinity for penicillin, thereby
conferring resistance to penicillin.
 PBP-2a is encoded by mecA, a gene located on the S.
aureus chromosome, which resides within the mobile genomic
island SCCmec (staphylococcal cassette chromosome mec). In
approximately 20 years, methicillin resistance became endemic in
the U.S., reaching 29 percent of hospitalized S. aureus-infected
patients.

37.  In 1967, strains of S. pneumoniae also became resistant to penicillin. By
1999, the percentage of cases associated with antibiotic-resistant
pneumococcus had tripled compared to 1979, reaching 14.4 percent in
South Africa.
 In 1976, beta lactamase-producing gonococci were isolated in England
and the U.S. Rapid spread of gonococcus resistance followed and in the
10-year period after the first introduction of penicillin to treat gonorrhea,
the prevalence of gonococcal penicillin-resistant strains reached its peak,
particularly in Asia.
 Furthermore, in 1983, a large outbreak of resistant non-beta-lactamase
producing gonococcus affected Durham city in North Carolina (U.S.).
Resistance of these strains was chromosomally-mediated, due to the
emergence of mutations that modified the penicillin target PBP2 and
expression of drug efflux pumps systems.
 Together, these events led to the prohibition of penicillin use as the first-
line drug for gonococcus treatment in most parts of the world.

38.  Another group of bacteria with high rates of penicillin resistance
is the Enterobacteriaceae, of which several strains are intrinsically
aminopenicillin-resistant, particularly among E. coli species.
Between 1950 and 2001, approximately two-thirds of E.
coli causing human diseases were ampicillin-resistant in the U.S.,
and the rate of aminopenicillin resistance is still on the rise.
 The development of resistance went hand in hand with the
introduction of new generations of penicillin into clinical practice.
More than 150 antibiotics have been found since the discovery of
penicillin, and for the majority of antibiotics available, resistance
has emerged.
 Moreover, the recent rise of multi/pan-drug resistant strains has
correlated with enhanced morbidity and mortality. Overall,
ineffectiveness of the antibiotic treatments to “superbug”
infections has resulted in persistence and spread of multi-resistant
species across the globe. This represents a serious worldwide
threat to public health.

39.  Have excellent tissue penetration
 Bactericidal against sensitive strains
 Relatively non toxic
 Efficacious in the treatment of infection
 Inexpensive in comparison with other antibiotics
 Newer penicillin’s are resistant to stomach acid such as penicillin V or
have broader spectrum, such as ampicillin and amoxicillin

40.  Acid liability – most of these drug are destroyed by gastric acid
 Lack of activity against most gram negative microorganism
 Short duration of action
 Many patients experience GI upset
 Painful if given intramuscularly

41.  Penicillin has a small risk of toxicity. Compared to other biologically
active substances, clinicians can administer these drugs at relatively
high doses without harming patients.
 Estimates are that it would take 5g/kg body weight intravenously to
cause convulsions in a patient. However, penicillin can cause local
toxicity due to high dose injections at sensitive sites such as the anterior
chamber of the eye or the subarachnoid space.
 There are reports that pure preparations of penicillin cause no harm to
the lungs and veins. Other reports indicate that topical penicillin can
prevent coagulation in dental cavities

42.  Penicillin is one of the most commonly used antibiotics globally, as it
has a wide range of clinical indications. Penicillin is effective against
many different types of infections involving gram-positive cocci, gram-
positive rods (e.g., Listeria), most anaerobes, and gram-negative cocci
(e.g., Neisseria).
 Importantly, certain bacterial species have obtained penicillin resistance,
including enterococci. Enterococci infections now receive treatment
with a combination of penicillin and streptomycin or gentamicin.
 Certain gram-negative rods are also resistant to penicillin due to
penicillin’s poor ability to penetrate the porin channel. However, later
generations of broad-spectrum penicillins are effective against gram-
negative rods.

43.  Second-generation penicillins (ampicillin and amoxicillin) can also
penetrate the porin channel, making these drugs effective
against Proteus mirabilis, Shigella, H. influenzae, Salmonella, and E.
coli.
 Third-generation penicillins such as carbenicillin and ticarcillin are
also able to penetrate gram-negative bacterial porin channels.
 Fourth-generation penicillins such as piperacillin are effective against
the same bacterial strains as third-generation penicillins as well
as Klebsiella, enterococci, Pseudomonas aeruginosa, and Bacteroides
fragilis.

44.  Patel, A. H. Industrial microbiology,2007. Rajiv Beri for Macmillan India Ltd.
New Delhi.
 Peter F. Stanbury, Allan Whitaker, Stephen J. Hall. Principles of Fermentation
Technology, 1997. Butterworth- Heinemann Ltd, Oxford
 Friedland IR, McCracken GH. Management of infections caused by antibiotic-
resistant Streptococcus pneumoniae. N Engl J Med. 1994 Aug 11;331(6):377-82.
 2.Herman DJ, Gerding DN. Antimicrobial resistance among
enterococci. Antimicrob Agents Chemother. 1991 Jan;35(1):1-4.
 3.Spratt BG, Cromie KD. Penicillin-binding proteins of gram-negative
bacteria. Rev Infect Dis. 1988 Jul-Aug;10(4):699-711.
 4.Perry CM, Markham A. Piperacillin/tazobactam: an updated review of its use
in the treatment of bacterial infections. Drugs. 1999 May;57(5):805-43.
 5.Fisher JF, Mobashery S. Constructing and deconstructing the bacterial cell
wall. Protein Sci. 2020 Mar;29(3):629-646.