Penicillin production by Likhith K

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.

1. Production of penicillin BY LIKHITH K B i SEP – 2021 Dept of Biotechnology St Aloysius College Mangaluru , Karnataka

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

3. Introduction ‘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. History 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. Structure 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

8. Mode of action 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.

9. 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 .

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

11. Outline of production

12. Production of penicillin 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

13. 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

14. Some high yielding strains of P . chrysogenum 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

15. 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 .

16. Sporulation medium- Moyer and Coghill (1946) Components g/L Glycerol 7.5 Cane molasses 7.5 Corn-steep liquor 2.5 MgSO 4. 7H 2 O 0.050 KH 2 PO 4 0.060 Peptone 5.00 NaCl 4.00 Fe-Tartrate 0.005 CuSO 4 -5H 2 0.004 Agar 2-50 Distilled water to make 1.0 ltr

17. 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 , T he 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.

18. Media 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.

19. 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

20. 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

21. Fermentation condition Penicillin production is an aerobic process and therefore, a continuous supply of O 2 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.

22. Phases in penicillium fermentation 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.

23. 4)extraction and purification 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

24. 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

25. 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

26. 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

27. Administration 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.

28. Adverse Effects 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

29. Contraindications 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. 

30. Penicillin Resistance: First Signs, Progression and the Global Problem 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.

31. 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 SCC mec  (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.

32. 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.

33. 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.

34. Advantages 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

35. Disadvantages 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

36. Toxicity 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

37. Conclusion 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.

38. 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 .

39. reference 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.

40. Thank you too much is always bad!!! Always consult a medical expert