c© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim10.1002/14356007.a02 467.pub2

Antibiotics 1

Antibiotics

Masaji Ohno, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan (Chap. 1, 2, 3 and 4)

Masami Otsuka, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan(Chap. 1, 2, 3 and 4)

Yoshinari Okamoto, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto,Japan (Chap. 1, 2, 3 and 4)

Morimasa Yagisawa, Japan Antibiotics Research Association, Tokyo, Japan (Chap. 1, 2, 3 and 4)

Shinichi Kondo, Institute of Microbial Chemistry, Tokyo, Japan (Chap. 1, 2, 3 and 4)

Heinz Oppinger, Hoechst Aktiengesellschaft, Frankfurt, Germany (Chap. 5, 6 and 7)

Hinrich Hoffmann, Hoechst Aktiengesellschaft, Frankfurt, Germany (Chap. 5, 6 and 7)

Dieter Sukatsch, Hoechst Aktiengesellschaft, Frankfurt, Germany (Chap. 5, 6 and 7)

Leo Hepner, L. Hepner and Associates, Ltd., London, United Kingdom (Chap. 8)

Celia Male, L. Hepner and Associates, Ltd., London, United Kingdom (Chap. 8)

1. Introduction . . . . . . . . . . . . . . 21.1. General Definition . . . . . . . . . . 21.2. Historical Development and

Classification . . . . . . . . . . . . . . 21.3. Nomenclature . . . . . . . . . . . . . 42. Chemotherapeutic Use of

Antibiotics . . . . . . . . . . . . . . . . 42.1. Microbial Pathogens . . . . . . . . . 42.2. Tumor Cells . . . . . . . . . . . . . . . 42.3. Chemotherapeutic Uses . . . . . . . 42.4. Use in Agriculture . . . . . . . . . . . 42.5. Units . . . . . . . . . . . . . . . . . . . 52.6. Analysis . . . . . . . . . . . . . . . . . 53. Classification of Antibiotics . . . . 53.1. β-Lactams . . . . . . . . . . . . . . . . 53.1.1. Natural Penicillins . . . . . . . . . . . 53.1.2. Semisynthetic Penicillins . . . . . . . 73.1.3. Natural Cephalosporins . . . . . . . . 73.1.4. Semisynthetic Cephalosporins . . . . 103.1.5. Cephamycins . . . . . . . . . . . . . . 113.1.6. 1-Oxacephems . . . . . . . . . . . . . . 133.1.7. β-Lactamase Inhibitors . . . . . . . . 143.1.8. Penems . . . . . . . . . . . . . . . . . . 143.1.9. Carbapenems . . . . . . . . . . . . . . 153.1.10. Monocyclic β-Lactams . . . . . . . . 163.2. Tetracyclines . . . . . . . . . . . . . . 163.3. Anthracyclines . . . . . . . . . . . . . 183.4. Aminoglycosides . . . . . . . . . . . . 183.5. Nucleosides . . . . . . . . . . . . . . . 233.5.1. N-Nucleosides . . . . . . . . . . . . . . 243.5.2. C-Nucleosides . . . . . . . . . . . . . . 243.5.3. Carbocyclic Nucleosides . . . . . . . 26

3.6. Macrolides . . . . . . . . . . . . . . . . 263.6.1. 12-Membered Ring Macrolides . . . 263.6.2. 14-Membered Ring Macrolides . . . 263.6.3. 16-Membered Ring Macrolides . . . 273.6.4. Polyenes . . . . . . . . . . . . . . . . . 283.7. Ansamycins . . . . . . . . . . . . . . . 283.8. Peptides . . . . . . . . . . . . . . . . . 293.9. Enediynes . . . . . . . . . . . . . . . . 323.10. Other Important Antibiotics . . . . 334. Antibiotic Resistance . . . . . . . . . 345. Fermentation . . . . . . . . . . . . . . 355.1. Screening . . . . . . . . . . . . . . . . 355.2. Selection, Mutation, and

Maintenance of Strains . . . . . . . 365.3. Process Development Leading to

Large-Scale Production . . . . . . . 365.4. Fermentation Technology . . . . . . 395.4.1. Maintenance of the Strain and

Production of Inoculum . . . . . . . . 395.4.2. Treatment Before and During

Fermentation . . . . . . . . . . . . . . . 406. Isolation and Purification of Anti-

biotics; Quality Specifications . . . 426.1. Isolation . . . . . . . . . . . . . . . . . 426.2. Purification Techniques, Sterile

End Products, Official Regulations 457. Analytical Measurements and

Quality Control . . . . . . . . . . . . 467.1. Microbiological Analysis . . . . . . 467.2. Isotopically Labeled Antibiotics . 488. Economic Aspects . . . . . . . . . . . 499. References . . . . . . . . . . . . . . . . 50

2 Antibiotics

1. Introduction

1.1. General Definition

In 1942, Waksman defined antibiotics as chemi-cal substances produced by microorganisms andcapable of inhibiting the growth of microorgan-isms [20]. Great effort has been devoted to theworldwide search for new antibiotics, and nu-merous compounds possessing various biologi-cal activities, that is, antibacterial, antiviral, anti-fungal, antitumor, and enzyme-inhibiting activi-ties, have been discovered. These substances aremostly of microbial origin but are also semisyn-thetic or totally synthetic in some cases. Theyhave a wide variety of structural characteristics.The area defined by the term “antibiotics” istherefore expanding.

1.2. Historical Development andClassification

In 1877, Pasteur observed that saprophytic bac-teria inhibited the growth of pathogenic anthraxorganisms. His was the first scientific descriptionof the antagonism phenomenon. Production ofa certain metabolic substance seemed to be re-sponsible for the inhibition. Pasteur suggestedthe therapeutic potential of this type of growthrepression. Vuillemin used the term “antibio-sis” to describe the inhibition of the growth ofone organism by another. The potential utilityof “bacteriotherapy” was recognized and enor-mous experimental efforts were made to inves-tigate the antagonism phenomenon.

In 1894, Metchnikoff reported the repressiveeffect of Pseudomonas on Vibrio cholerae. Fromthe culture of a Penicillium, Gosio isolated an an-tibacterial crystalline substance, mycophenolicacid, in 1896. Other results also demonstratedthe ability of various microbes to produce an-tibacterial substances [21].

It was in 1929 that Fleming observed that aculture of a Penicillium inhibited the growth ofbacteria [22]. He demonstrated the productionof an antibacterial substance in the culture brothand named it penicillin. Although he suggestedthe promising therapeutic utility of penicillin,none of the attempts to isolate penicillin weresuccessful and attention was not attracted for

the next decade. Instead, synthetic chemother-apeutics, such as sulfonamides, became objectsof general interest after the discovery of pron-tosil by Domagk in 1935 [23]. Only the outbreakof the Second World War in 1939 led to an in-tense worldwide search for drugs to treat infec-tions and wounds. Toward the end of the 1930s,Florey, Chain, and co-workers began to inves-tigate penicillin in the course of their systematicstudy of antibacterial substances. They demon-strated the marked activity and therapeutic valueof penicillin in 1940 [24]. The production ofpenicillin had until that time been unsatisfac-tory, and favorable conditions for the effectiveformation of the antibiotic were explored in theUnited States. An active culture of the penicillin-producing organism was sought and submergedfermentation was developed. The use of lactoseas a carbon source and the addition of cornsteepliquor to the nutrients were found effective. Irra-diation of the culture with X-rays or ultravioletlight produced mutant strains. These findings setthe stage for the industrial production of peni-cillin.

After the discovery of penicillin, Brotzubegan a search for antibiotic-producing organ-isms and examined a culture of Cephalospo-rium spp. isolated from the sea near a sewageoutlet in Sardinia. It secreted substances activeagainst gram-positive bacteria. In September of1948, Brotzu sent his organism to Abrahamat Oxford for detailed inspection. Several anti-biotics were isolated from the culture and namedcephalosporin [25]. Particular attention was at-tracted by cephalosporin C, crystallized from thecrude mixture of antibiotics, because of its lowtoxicity and its resistance to penicillinase.

Penicillins and cephalosporins, both of whichpossess a β-lactam ring as a structural char-acteristic, are designated β-lactam antibiotics.Extensive attempts to improve their antibacte-rial spectra through chemical modifications ledto the development of many kinds of semisyn-thetic penicillins and cephalosporins. Moreover,nontraditional β-lactams, namely penems, car-bapenems, and monobactams, have been disco-vered and commercialized [26 – 29].

Other novel antibiotics were discoveredamong the products of fungi, bacteria, and acti-nomycetes [30]. As a result of a search forwater-soluble and heat-stable substances whichwould be active against gram-negative bacte-

Antibiotics 3

ria, Waksman isolated actinomycin in 1940 andstreptomycin in 1944 from cultures of actino-mycetes. Various other antibiotics were isolatedfrom microbes in France, Germany, Japan, theUnited Kingdom, the United States, and othercountries.

A yellow substance showing antibacterialactivity was found in 1948 among productsof Streptomyces aureofaciens and was namedaureomycin. Terramycin was isolated from thefermentation broth of Streptomyces rimosus.The chemical and structural similarities of thetwo soon became apparent; they each havea linearly fused tetracyclic structure of six-membered rings. This parent skeleton is desig-nated “tetracycline,” and aureomycin and terra-mycin are now called chlortetracycline and oxy-tetracycline, respectively.

In 1950, rhodomycin was isolated from theculture broth of Streptomyces purpurascens byBrockmann and his co-workers. Structural anal-ysis disclosed that rhodomycin is a glycoside,combining an amino sugar and a 7,8,9,10-tetra-hydro-5,12-naphthacenequinone moiety. Struc-turally similar antibiotics have since been disco-vered, and the generic name “anthracycline” hasbeen assigned. The aglycone of anthracycline iscalled anthracyclinone. Daunorubicin and dox-orubicin are representative anthracycline anti-tumor antibiotics. Aclarubicin possesses threesugars and is of interest because of its low toxi-city.

Streptomycin is used for infections of gram-positive and gram-negative bacteria and as aspecific medicine for tuberculosis. Kanamycin,discovered by Umezawa in 1957, is especiallyeffective against resistant bacteria. Paromo-mycin, Spectinomycin, and ribostamycin wereisolated later. These antibiotics are called ami-noglycosides because their structural units areamino sugars, sugars, and amino acids. They arewater-soluble and basic in nature. The mecha-nism of resistance to aminoglycosides has beenclosely investigated and derivatives for use ac-tive against the resistant bacteria have been de-veloped.

The first nucleoside antibiotic, cordycepin,was isolated in 1950. Various nucleoside anti-biotics of unusual structure have subsequentlybeen isolated and are important as antibacteri-als, antineoplastics, and agricultural chemicals.

Macrolides are macrocyclic lactones towhich sugars are attached. Various clinicallyimportant antibiotics are macrolides. Erythro-mycin, isolated in 1952, has two sugars con-nected to different sites on the 14-memberedring aglycone. Dimethylamino sugars are of-ten found in macrolide antibiotics. Macrolidesare classified as 12-, 14-, or 16-membered ringmacrolides.

Ansamycin has an aliphatic “ansa” bridgespanning two nonadjacent positions of the aro-matic system. Rifamycin is a representativeansamycin possessing a 1,4-naphthoquinonemoiety. Polyene antibiotics have a conjugatedolefinic structure in the macrocyclic lactonemoiety, as is the case with amphotericin B,and sometimes lack the amino sugar moiety.Polyenes are produced mainly by Streptomycesand show antifungal activity.

Actinomycin is a peptide antibiotic effectiveagainst tumor cells as well as bacteria. Peptideantibiotics, one of the major antibiotics groups,possess diverse activities, i.e., antibacterial, an-tifungal, and antitumor activities. Their struc-tures are varied and feature complicated modesof connection of unusual amino acids as envis-aged in bleomycin. Ring peptides, linear pep-tides, lactonic peptides, and peptides containinghydroxy acids have been isolated. The group in-cludes actinomycins, gramicidins, polymyxins,and colistins.

Enediyne antibiotics have a medium-sizedring containing a double bond and two triplebonds that generate carbon radicals to induceDNA damage. Neocarzinostatin was the firstenediyne discovered in 1957. More recently,calicheamicin and esperamicin were isolated.

Chloramphenicol was first isolated as a prod-uct of Streptomyces venezuelae; it showed abroad antimicrobial spectrum. Chloramphenicolis an unusual natural product because it pos-sesses both chloro and nitro groups in its struc-ture. It is the only antibiotic produced commer-cially by an entirely chemical synthesis.

In addition to the antibiotics described above,various unclassified antibiotics have been iso-lated. The most important antibiotics and deriva-tives are discussed in this article. Some repre-sentative compounds that have recently been in-troduced in clinics are also described.

4 Antibiotics

1.3. Nomenclature

In principle, it is the privilege of the discoverer toname to his or her new antibiotic. Usually anti-biotics are named after the producing organismsor some aspect of their chemical and biologicalnature. In accordance with the suggestions of theNomenclature Committee of the American So-ciety of Microbiology [31], names of antibioticsshould be based on (a) the family to which theantibiotic belongs, (b) the chemical structure ofthe compound, or (c) some property of the an-tibiotic. If, for some reason, a name cannot begiven to the new antibiotic, a code designationmay be given.

2. Chemotherapeutic Use ofAntibiotics

2.1. Microbial Pathogens

Microorganisms that can be treated bychemotherapy include bacteria, fungi (→ Anti-mycotics), viruses, rickettsia, and protozoa (→Chemotherapeutics).

Outside the cytoplasmic membrane, bacteriahave a rigid shell called the cell wall that is notseen in mammalian cells. The main constituentof the cell wall is peptidoglycan, a cross-linkedstructure of long parallel chains of polysaccha-rides and short peptide chains. Bacteria are sep-arated into two classes based on the results of aGram’s stain. Gram-positive bacteria hold thecolor of the primary stain and gram-negativeones are decolorized and are stained by the coun-terstain. The structure and constituents of the cellwalls of gram-positive and gram-negative bac-teria are slightly, but distinctly, different.

Some antibiotics are effective only againstgram-positive bacteria whereas others are ac-tive against gram-negative ones. Streptomycinand kanamycin are effective against mycobacte-ria as well as gram-positive and gram-negativebacteria. Chloramphenicol and tetracyclines arebroad-spectrum antibiotics active against notonly the usual bacteria but also rickettsia.

2.2. Tumor Cells

Certain antibiotics are effective for clinical treat-ment of cancer. Cancer cells function abnor-

mally, escaping growth regulation. Antibioticsoccupy an important position among the vari-ous agents for cancer chemotherapy. Doxoru-bicin, daunorubicin, mitomycin C, bleomycin,actinomycin D, and neocarzinostatin are usedclinically. They interact with DNA to inhibit po-lymerases of DNA and RNA, or to cause DNAstrand breakage. Normal cells are also damagedto some extent; the selective toxicity is gener-ally based on the unusually rapid multiplicationof the tumor cells. Some of these antibiotics alsopossess anti-neoplastic activity because they in-hibit the synthesis of DNA.

2.3. Chemotherapeutic Uses

Chemical and bacteriological diagnoses are es-pecially important for successful chemotherapybecause the choice of drug depends primarilyon the sensitivity of the microorganism to thedrug. The antimicrobial activity of an antibi-otic is expressed as the minimum inhibitoryconcentration (MIC) measured by the dilutionmethod. Antibiotics are administered by hypo-dermic, intramuscular, or intravenous injections,or as internal medicines. For external applica-tions, they are given according to the nature ofthe antibiotic and the characteristics of the dis-ease. Doses large enough to maintain a suffi-cient drug concentration in the blood and tis-sues are prescribed. Various undesirable side ef-fects of antibiotics have been reported. One ofthe serious side effects is the allergic reaction topenicillins. Oto- and nephrotoxicity result fromthe long-term use of aminoglycosides in quan-tity. Aplastic anemia caused by chloramphenicolalso has been reported. Antitumor and antiviralantibiotics are generally highly toxic.

2.4. Use in Agriculture

Although antibiotics were originally developedfor use against microbial diseases in humans,they are also applicable to agriculture. Severalantibiotics are used in the treatment of animaland plant diseases. Kasugamycin and blastcidinS are effective against rice blast disease. Poly-oxins are selectively effective against certain

Antibiotics 5

species of phytopathogenic fungi. Antibioticsare used for veterinary purpose as therapeuticagents to prevent breeding disease and fodderadditives to stimulate growth of animal. How-ever, there is the danger of developing a resis-tance resulting from the veterinary use of anti-biotics. WHO recommended to reduce the useof antibiotics as an additive in animal fodder be-cause it may raise the number of drug-resistantbacteria in animal meat. Therefore, worldwideefforts are directed at avoiding the use of thetherapeutically important antibiotics, particu-larly the penicillins and the tetracyclines, as feedadditives. Instead, these antibiotics are used onlyfor genuine veterinary treatment in compliancewith certain controls and quarantine guarantees.

2.5. Units

The production, isolation, and processing ofcommercial products require careful control forall pharmaceuticals. Because of the extremelyhigh sensitivity and the danger of diminishedactivity, this consideration has always been par-ticularly important.

The Oxford unit (O. U.) is defined as theamount of penicillin that just prevents the growthof a certain Staphylococcus aureus species. Verypure crystalline penicillin salts generally haveconstant biological activities and the Oxford unithas been replaced by the international unit: 1 mgof pure benzylpenicillin sodium contains 1670O. U.; the O. U. specific to this salt was declaredto be the international unit (I. U., usually ab-breviated U). Conversely, 0.6 µg of benzylpeni-cillin sodium has the activity of 1 I. U. Becausethe biological activity comes from the penicillinnucleus, the change to another cation leads toa change in activity proportional to the molec-ular mass. This change can be calculated. Theactivities of the chief penicillin salts are:

benzylpenicillin sodium 1670 U/mgbenzylpenicillin potassium 1598 U/mgbenzylpenicillin procaine 1011 U/mgpenicillin-2-hydroxyprocaine 1008 U/mgpenicillin-N,N′-dibenzyl-ethyl-enediamine

1213 U/mg

penicillin-N-ethylpiperidine 1328 U/mg.

The mass of 1 U, for benzylpenicillin sodium,0.6 µg, is extremely small. The following largerunits of mass are used in production and trade:

1 Mega U = 1×106 I. U.= 600 mg benzylpenicillin sodium= ca. 1 g benzylpenicillin procaine

1 Mio Mega U = 1×1012 I. U.= 600 kg benzylpenicillin sodium= ca. 1 t benzylpenicillin procaine

The activities of some older penicillins aregiven in I. U.

phenoxymethyl-penicillin:

1 mg free acid 1699 U

phenethicillin: 1 mg d-potassium salt 1476 U1 mg l-potassium salt 1470 U

penicillin O: 1 mg potassium salt 1612 U

All other penicillins that are used therapeuti-cally can be made very pure and the preparationsare dosed and traded in mass units (µg, mg, g,kg).

2.6. Analysis

The practical determination of active substancesin penicillins and other antibiotics can be dividedamong three types of methods [25]:

1) Microbiological testing (see Chap. 7).2) Determination of the contents by chemical or

enzymatic conversion followed by a physicalmethod, such as colorimetry.

3) Purely physical methods, such as UV or IRabsorption.

3. Classification of Antibiotics

3.1. β-Lactams

The β-lactam group includes natural peni-cillins, semisynthetic penicillins, naturalcephalosporins, semisyntheric cephalosporins,cephamycins, 1-oxacephems, β-lactamase in-hibitors, penems, carbapenems, nocardicins,and monobactams.

3.1.1. Natural Penicillins

Penicillin was discovered in 1929 by Fleming[22]. At first it was obtained as a mixture ofseveral similar compounds, but these were later

6 Antibiotics

separated from each other. The β-lactam struc-ture of penicillin was proposed by Abraham andChain and supported by Woodward, but it wasopposed by those who believed in the alterna-tive thiazolidine-oxazole structure [33]. The β-lactam structure was finally established by anX-ray crystallographic analysis performed byHodgkin and Low [34].

Several penicillins have been isolated fromthe fermentation broths of Penicillium nota-tum or P. chrysogenum. Of these, benzylpeni-cillin (penicillin G) shows good stability, activ-ity, and rate of production by microorganisms.Benzylpenicillin benzathine was developed as adilatorily acting benzylpenicillin that maintainsan effective serum concentration for 2 days aftera single intramuscular injection. Total synthesisof penicillin V was achieved by Sheehan andHenery-logan in 1957 [35]. Biogenic synthe-ses of penicillin – cephalosporin antibiotics alsohave been reported [36, 37].

Name R

Benzylpenicillin(Penicillin G) [61-33-6]

Phenoxymethylpenicillin(Penicillin V) [87-08-1]

Phenethicillin[147-55-7]

Methicillin[61-32-5]

Ampicillin[69-53-4]

Amoxicillin[26787-78-0]

Ciclacillin[3485-14-1]

Piperacillin[61447-96-1]

Aspoxicillin[63358-49-6]

Mecillinam[32887-01-7]

Benzylpenicillin benzathine [41372-02-5]

Antibiotics 7

3.1.2. Semisynthetic Penicillins

Several limitations have become apparent con-cerning the antibiotic activity of benzylpeni-cillin. This drug is not very active against gram-negative bacteria; it is inactivated by penicil-linase produced by resistant organisms, and itis not suitable for oral administration becauseit breaks down under acidic conditions. Peni-cillins having variously altered side chains havebeen made by adding appropriate precursors tothe fermentation [33]. Various penicillins havebeen obtained biosynthetically. Among these isphenoxymethylpenicillin (penicillin V) the firstused by oral administration. In contrast, 6-ami-nopenicillanic acid (6-APA) can be prepared byeither enzymatic or chemical means. Penicillinamidase or penicillin acylase cleaves the sidechain of penicillin to produce 6-APA. The amidebond of the side chain is also efficiently cleavedby treatment with phosphorus pentachloride [25,p. 27]. Penicillins with modified side chains havebeen synthesized from 6-APA in order to im-prove the antibacterial spectra and increase thestability against penicillinase [33, p. 59]. Ampi-cillin is active against gram-negative bacteria.Ampicillin and amoxicillin are suitable for oraladministration. Methicillin was developed as β-lactamase-resistant penicillin. However, emer-gence of methicillin-resistant Staphylococcusaureus (MRSA), first identified in 1961, is a cur-rent major problem. Mecillinam has an unusualamidino side chain and is relatively stable andeffective against gram-negative bacteria.

6-Aminopenicillanic acid (6-APA) [551-16-6]

Modification of the carboxyl group has beenfound to be effective for the purpose of oral ad-ministration, and penicillin esters talampicillin,bacampicillin, lenampicillin, and pivmecillinamhave been developed [33, p. 59]. These are ab-sorbed and hydrolyzed by the small intestine torelease free acids of the parent penicillins.

Name R1 R2

Talampicillin

[47747-56-8]

Bacampicillin

[50972-17-3]

Lenampicillin

[86273-18-9]

Pivmecillinam

[32886-97-8]

3.1.3. Natural Cephalosporins

The fermentation broth of Cephalosporiumspp. isolated by Brotzu contained severalantibiotics: cephalosporin P, penicillin N, andcephalosporin C. Cephalosporin P was shown tobe an acidic steroidal substance. CephalosporinC was active against gram-negative bacteria, re-sistant to β-lactamase, and much less toxic thanpenicillin. The chemical structure of cephalo-sporin C was determined by Abraham and

8 Antibiotics

Name R1 R2 Name R1 R2

Cephalosporin C

[61-24-5]

Cefmenoxime

[65085-01-0]

Cefalexin

[15686-71-2]

Ceftizoxime

[68401-81-0]

Cefaclor

[53994-73-3]

Cefotaxime

[63527-52-6]

Cefroxadine

[51762-05-1]

Cefuroxime

[55268-75-2]

Cefadroxil

[50370-12-2]

Ceftriaxone

[73384-59-5]

Cefalotin

[153-61-7]

Cefodizime

[69739-16-8]

Antibiotics 9

Name R1 R2 Name R1 R2

Cefsulodin

[62587-73-9]

Cefdinir

[91832-40-5]

Cefatrizine

[51627-14-6]

Cefixime

[79350-37-1]

Cefoperazone

[62893-19-0]

Ceftibuten

[97519-39-6]

Cefotiam

[61622-34-2]

Cefpiramide

[70797-11-4]

Cefazolin

[25953-19-9]

10 Antibiotics

Newton [25]. It consists of 7-aminocephalo-sporanic acid (7-ACA) and D- a-aminoadipicacid. Woodward and his co-workers synthesizedcephalosporin C in a fully stereospecific manner[38].

7-Aminocephalosporanic acid (7-ACA) [957-68-6]

3.1.4. Semisynthetic Cephalosporins

Because the antibacterial activity of cephalo-sporin C itself is relatively low, the develop-ment of a more active derivative is desirable.The phosphorus pentachloride method has beenapplied to the cephalosporin system to produce7-ACA in high yield [25, p. 27]. The 3′-acetoxygroup of cephalosporin is easily replaced byvarious nucleophiles [25, p. 134]. Modificationof the 7-amino group and the 3′ group makepossible the various cephalosporin derivatives[33, p. 59]. Cefazolin is active against gram-negative bacteria. Cefuroxime, cefotaxime, andceftizoxime have a methoxyimino group and a2-aminothiazole ring in common and are resis-tant to β-lactamase. Cefoperazone is particu-larly active against Pseudomonas. All of theseare used by injection. On the other hand, severalcephalosporins are used only by oral administra-tion. These include cephalexin, cefadroxil, cefa-clor, cefroxadine, and cefatrizine.

Cephalosporins are classified into four gen-erations according to their antibacterial proper-ties. The first generation includes cefalexin, ce-fadroxil, cefotiam, and cefazolin. β-Lactamasestability is improved in the second-generationcephalosporins that includes cefaclor, cefurox-ime, and cefixime. The third generation includescefsulodin, cefoperazone, cefotaxime, ceftria-zone, ceftibuten, and ceftazidime. Frequent useof the third generation resulted in the emergenceof MRSA. The fourth generation includes cef-pirome and cefitime.

Cephalosporin esters are also developed fororal administration. These are cefotiam hexetil,cefteram pivoxil, cefditren pivoxil, cefcapenepivoxil, cefpodoxime proxetil, and cefuroximeaxetil.

Name StructureCefpirome

[84957-29-9]

Cefepime

[88040-23-7]

Cefopiran

[113359-04-9]

Ceftazidime

[72558-82-8]

Antibiotics 11

Name Structure Name Structure

Cefotiamhexetil

[95761-91-4]

Cefterampivoxil

[82547-81-7]

Cefditorenpivoxil

[117467-28-4]

Cefcapenepivoxil

[105889-45-0]

Cefpodoximeproxetil

[87239-81-4]

Cefuroximeaxetil

[64544-07-6]

Cephalosporins are also obtainable via thering expansion reaction of penicillin sulfox-ide first devised by Morin [25, p. 183]. Thus,cephalexin is produced by chemical conversionof phenoxymethylpenicillin or benzylpenicillin.

3.1.5. Cephamycins

Substances similar to cephalosporin C werefound among the products of various strepto-mycetes and were characterized by the pres-ence of a 7α-methoxy group. They are namedcephamycins after their cephalosporin skeleton

12 Antibiotics

and their production by streptomycetes [39],[29, vol. 1, p. 199]. They are strongly resis-tant to β-lactamase and effective against gram-negative bacteria and bacteria that have acquiredresistance to penicillins and cephalosporins.Semisynthetic cephamycins with improved ac-tivities are obtained by chemical transforma-tions. These include cefmetazole, cefotetan,cefminox, and cefbuperazone.

Name StructureCephamycin C

[34279-51-1]

7-Aminocephamycinoic acid (7-ACMA)

[34279-51-1]

Cefmetazole

[56796-20-4]

Cefotetan

[69712-56-7]

Name StructureCefminox

[84305-41-9]

Cefbuperazone

[76610-84-9]

Chemical modification of cephamycins re-quires special devices for the following reasons.7-Aminocephamycinoic acid (7-ACMA), whichcorresponds to the 7-ACA of cephalosporins, isnot easily isolated because of its instability.

It has methoxy and amino groups on the samecarbon atom of the β-lactam ring and the elim-ination of the protonated amino group is quitefacile, because of the electron-donating natureof the methoxy group. Moreover, the usual phos-phorus pentachloride method cannot be appliedto the side chain cleavage of cephamycin C be-cause a strong N−P bond is formed by the re-action of the carbamate moiety of cephamycinC with phosphorus pentachloride [40]. Instead,exchange of the α-aminoadipoyl side chain foranother acyl group is achieved by treating thefully protected cephamycin C with the appro-priate acyl chloride in the presence of a neutralacid scavenger. This is followed by the simul-taneous removal of the amino protective groupand the α-aminoadipoyl group [41]. The side-chain transformation is also effected using anacyl chloride and partially hydrated molecularsieves [42] (see top of page 13, scheme 1).

Chemical conversion of cephamycin into 7-ACMA ester has been reported [43] (see top ofpage 13, scheme 2).

Antibiotics 13

scheme 1

scheme 2

3.1.6. 1-Oxacephems

In addition to the modification of the side chainsof natural β-lactam antibiotics, totally or par-tially synthetic nuclear analogs of penicillins andcephalosporins have been explored extensively[28 33, p. 59]. In 1974 Wolfe reported the first1-oxacephem derived from penicillin, but its an-tibacterial activity remains unknown because theamino and carboxy protective groups have notbeen removed. Racemic 1-oxacephalothin, syn-thesized by Christensen and his co-workers inthe same year, was found to be antibacteriallyactive, suggesting that the sulfur atom is not al-ways necessary for the expression of antibioticactivity. Nagata developed latamoxef which ex-hibits strong activity against pathogenic anaer-obes, such as Bacteroides fragilis, as well asgram-negative bacteria, including Pseudomonas[29, vol. 2, p. 1]. It is completely stable againstvarious β-lactamases and has low toxicity. Ahigh plasma-peak level and long duration aremaintained. Latamoxef is a nuclear analog ofcephamycin; it is produced on an industrial scaleby a totally chemical process starting with epi-penicillin S-oxide [44, 45]. Flomoxef is anotherclinically used 1-oxacephem that shows wide ac-

tivity against gram-positive and gram-negativebacteria [46].

Name Structure

1-Oxacephem (Wolfe, 1974)

[54997-17-0]

1-Oxacephalothin

[54214-83-4]

Latamoxef

[64952-97-2]

14 Antibiotics

Name StructureFlomoxef

[99665-00-6]

3.1.7. β-Lactamase Inhibitors

In the course of screening the substances inhibit-ing β-lactamase, which is responsible for bacte-rial resistance to penicillins and cephalosporins,a potent β-lactamase inhibitor, clavulanic acid,was isolated from Streptomyces clavuligerus[47]. Clavulanic acid is characteristic for its 1-oxadethiapenem ring system and the lack of theside chain at position 6. The antibiotic activityof clavulanic acid is not strong, but it has a broadantibacterial spectrum. Instead, it is effectivesynergistically when used with β-lactamase-sensitive penicillins and cephalosporins againstβ-lactamase-producing organisms. Clavulanicacid is used in combination with amoxicillin.Sulbactam is a semisynthetic inhibitor of β-lactamase having a penam sulfone structure[48]. Combinations of sulbactam–ampicillin,sulbactam–cefoperazone are clinically used.Sultamicillin is an ester-linked chimeric drugcomprising sulbactam and ampicillin releasingthe two constituents when administered [49].Tazobactam is a derivative of sulbactam usedin combination with piperacillin [50].

Name StructureClavulanicacid

[58001-44-8]

Sulbactam

[68373-14-8]

Name StructureTazobactam

[89786-04-9]

Sultamicillin

[76497-13-7]

3.1.8. Penems

The penem ring system has not been foundin nature; it has been designed artificially byWOODWARD [26, p. 167], [28 29, vol. 2, p.315]. It is widely accepted that the antibacte-rial activity of β-lactam antibiotics is based ontheir ability to acylate enzymes. In penicillins,the rigid, nonplanar bicyclic system enhancesthe reactivity of the β-lactam ring by dimin-ishing the delocalization of the unshared elec-tron pair of the amide nitrogen onto the ad-jacent carbonyl group. On the other hand, incephalosporins, where the β-lactam nitrogen isbonded almost planar, the double bond of thesix-membered ring interacts with the unsharedelectrons of the β-lactam nitrogen, diminish-ing the delocalization to the amide carbonyl.Therefore, the β-lactam ring of cephalosporinsis cleaved easily. Penems combine the twostructural elements, the five-membered ring andthe double bond. 6-Acylaminopenem-3-carbox-ylic acids, 6-unsubstituted penem-3-carboxyl-ic acids, and 6-alkylpenem-3-carboxylic acidshave been synthesized. Faropenem is a clinicallyused penem active against penicillin-resistantpneumonococcus [51].

Antibiotics 15

Name Structure6-Acylaminopenem-3-carboxylicacid

Penem-3-carboxylic acid

6-Alkylpenem-3-carboxylic acid

Faropenem

[106560-14-9]

3.1.9. Carbapenems

Cabapenems are a family of antibiotics havingthe 1-azabicyclo[3.2.0]hept-2-ene system [2729, vol. 2, p. 227]. The first carbapenem an-tibiotic, thienamycin, was discovered at Merckin 1976 among the fermentation products ofStreptomyces cattleya [53]. Antibiotics of thistype have been isolated one after another in thesearch for inhibitors of bacterial cell wall syn-thesis and β-lactamase. Imipenem, a semisyn-thetic derivative of thienamycin, is the first car-bapenem brought into clinical use [54]. It isused in combination with cilastatin, which isan inhibitor of dehydropeptidase-I that inac-tivates imipenem. Cilastatin also suppress thenephrotoxicity of imipenem. Meropenem andbiapenem are currently developed carbapenems[55, 56]. Panipenem is used with betamipron,which reduces the nephrotoxicity of panipenemby suppressing the transportation into kidney[57].

Name StructureThienamycin

[159995-64-1]

Imipenem

[64221-86-9]

Cilastatin

[82009-34-5]

Meropenem

[96036-03-2]

Biapenem

[12410-24-4]

Panipenem

[87726-17-8]

16 Antibiotics

Name StructureBetamipron

[3440-28-6]

3.1.10. Monocyclic β-Lactams

A mutant strain of Escherichia coli showing spe-cific supersensitivity to β-lactam antibiotics hasbeen and used to isolate nocardicins from No-cardia uniformis by a screening procedure [58].The nocardicin structure has been elucidated byspectroscopic analysis and chemical degrada-tion. The noncardicins are monocyclic β-lactamantibiotics [27], [29, vol. 2, p. 165], [52, p. 281].The clinical application of was not successfulbecause of hepatotoxicity.

Sulfazecin was isolated in 1981 as a prod-uct of Pseudomonas acidophila by screeningusing organisms highly sensitive to β-lactams[59]. The structure was shown to be a mono-cyclic β-lactam. In the same year, anotherresearch group independently reported on aseries of monocyclic β-lactams produced byAgrobacterium, Chromobacterium, and Glu-conobacter, including a compound identicalto sulfazecin [60]. A name “monobactam”was proposed for compounds characterized bythe 3-acylamino-2-oxoazetidine-1-sulfonic acidgroup. In monobactams, the β-lactam ring pre-sumably is activated by the electronic effect ofthe sulfonate moiety alone, in contrast to the caseof penicillins and cephalosporins. Because theantibacterial activity of sulfazecin is not satis-factory, many derivatives have been synthesizedchemically [29, vol. 3, p. 339]. Among them,aztreonam, synthesized from threonine, hasbeen found highly effective [61]. Carumonam isalso a β-lactamase-resistant nomobactam activeagainst gram-negative bacteria including Pseu-domonas [62].

Name StructureNocardicin A

[39391-39-4]

Sulfazecin

[77912-79-9]

Aztreonam

[78110-38-0]

Carumonam

[87638-04-8]

3.2. Tetracyclines

The first tetracycline aureomycin (chlortetra-cycline) was was found in the culture broth ofStreptomyces aureofaciens by Duggar [63].

The linear four-ring-system skeleton is char-acteristic of the tetracyclines and has given thewhole group its name. The strongly conjugatedsystem of keto and enol groups is of particularsignificance for the biological activity.

Antibiotics 17

The tetracyclines are bright yellow com-pounds, amphoteric, and with the exceptionof rolitetracycline and similarly constructedderivates insoluble in water at the isoelectricpoint. Their salts, e.g., hydrochlorides, are solu-ble in water and can be administered either par-enterally or orally, although the low pH of thesolution causes some problems in the latter in-stance.

Chlortetracycline (aureomycin) shows awider range of antibiotic activity compared withthe earlier antibiotics, penicillins, and strepto-mycins. Its activity covers gram-positive andgram-negative bacteria as well as Rickettsiaand Chlamydiae. Chlortetracycline has been re-placed by other tetracyclines in clinical use.

Tetracycline was first obtained from chlor-tetracycline by reductive dehalogenation in 1953

[143]. It was also obtained either by fermenta-tion of the chlortetracycline-producing organ-ism, Streptomyces aureofaciens, under condi-tions of chlorine limitation, or by fermentationof a mutant of the organism lacking the chlori-nating enzyme. Tetracycline is more stable thanchlortetracycline in aqueous solution. Its antimi-crobial activity is the same as that of chlortetra-cycline and oxytetracycline, but its serum con-centration after oral administration is consider-ably higher and more enduring.

Demethylchlortetracycline shows morepositive antimicrobial activity in vitro and pro-tective effect in vivo compared with tetracycline[144].

Oxytetracycline has had wide clinical use asa substitute for chlortetracycline, even after theintroduction of tetracycline [145].

18 Antibiotics

Doxycycline was synthetic compound show-ing antibacterial activity four times stronger thantetracycline against a variety of pathogens [146].Oral absorption of doxycycline is higher thanthat of tetracycline achieving higher tissue con-centration in tissue that is maintained longer.

Minocycline was also a synthetic com-pound active against tetracycline-resistant bac-teria showing higher activity compared withtetracycline against a variety of pathogens [147].Minocycline is widely used by oral administra-tion and by drip infusion for serious infections.

3.3. Anthracyclines

Anthracycline antibiotics are structurally gly-cosylated tetracyclines. The aglycone is char-acterized by the fused cyclohexane–benzene–p-quinone–benzene system having hydroxyland/or methyl substituents. Although anthra-cyclines show antibacterial activity, they havenot been used as antibiotics because of theirrelatively high toxicity and strong side effects.The antitumor activity of rhodomycin was disco-vered by Arcamone et al. in 1961 [64], and vari-ous antitumor anthracyclines were subsequentlyisolated [65]. Daunorubicin and doxorubicinare representative anthracyclines. Daunorubicinwas found in 1963 in the mycelium of Strepto-myces peucetius and the culture broth of Strep-tomyces coeruleorubidus, respectively [148]. Itwas the first anthracycline antibiotic clinicallyused for therapy of cancers, especially leukemia.Doxorubicin was found in the culture brothof Streptomyces peucetius var. cesius in 1967[149]. It shows stronger activity against a varietyof tumors and leukemia than daunorubicin, andits clinical application in the therapy of canceris wider than that of daunorubicin. Aclarubicinwas found by Umezawa in the culture broth ofStreptomyces galilaeus MA144-M1 in 1975 andevaluated its strong antileukemic activity andlow cardiac toxicity [150]. Pirarubicin is a tetra-hydropyranyl derivative of doxorubicin. Epiru-bicin, idarubicin, and amrubicin are currently inclinical use. Anthracyclines exert their effect byintercalating DNA [66].

3.4. Aminoglycosides

Waksman discovered the first useful aminogly-coside, streptomycin, in 1944 [67 – 71]. Afterwide use of penicillin, streptomycin, chloram-phenicol, and tetracycline, resistant organismsappeared in hospital patients. In 1957, staphylo-cocci and gram-negative organisms resistant toall the known antibiotic drugs caused serious in-fections; kanamycin was discovered at that timeby Umezawa and was introduced clinically.

More than 150 naturally occurring ami-noglycosides have been isolated from culturefiltrates of Streptomyces, Streptoverticillium,Nocardia, Micromonospora, Streptoalloteichus,Dactylosporangium, Saccharopolyspora, andother bacterial strains.

Most aminoglycoside antibiotics that are im-portant for chemotherapy contain 1,3- or 1,4-diaminocyclitols named actinamine, 2-deoxy-streptamine, fortamine, or streptidine. Amongthese naturally occurring aminoglycosides,kanamycin A, kanamycin B, ribostamycin, si-somicin, spectinomycin, streptomycin, tobra-mycin, gentamicins, neomycins (fradiomycins)are clinically used. Hygromycin B and desto-mycin A are used as animal anthelmintics.Kasugamycin is used for the prevention ofplant diseases. Among resistant bacteria ofclinical origin, the most important mecha-nism of resistance to aminoglycoside antibioticsis the inactivation by O-phosphorylation, O-nucleotidylation, or N-acetylation of specificsites of the antibiotic. The gene for these en-zymes is located on a plasmid. Organisms withresistance resulting from permeability barriersto drugs have been isolated, but ribosomal re-sistance to aminoglycosides is very rare in or-ganisms isolated clinically. Studies of the enzy-matic mechanism of resistance to aminoglyco-sides have been reviewed [73 – 76].

Semisynthetic aminoglycosides have beendeveloped based on the enzymatic mechanismof resistance. 3′-Deoxykanamycin A has beensynthesized and used to inhibit the growthof resistant strains having aminoglycoside-3′-phosphotransferase enzymes. Dibekacin (3′,4′-dideoxykanamycin B), synthesized from kana-mycin B, shows a strong activity not only againstresistant staphylococci and gram-negative or-ganisms but also against Pseudomonas [72].

Antibiotics 19

Name R1 R2 R3 R4 R5 R6 R7

Daunorubicin[20830-81-3]

OCH3 H OH H COCH3 OH A

Doxorubicin[23214-92-8]

OCH3 H OH H COCH2OH OH A

Rhodomycin A[1404-50-8]

OH H OH B CH2CH3 OH B

Rhodomycin B[1404-52-0]

OH H OH OH CH2CH3 OH B

Aclarubicin[57576-44-0]

OH H H CO2CH3 CH2CH3 OH C

Pirarubicin[72496-41-4]

OCH3 H OH H COCH2OH OH D

Epirubicin[56420-45-2]

OCH3 H OH H COCH2OH OH D

Idarubicin[20830-81-3]

H H OH H COCH3 OH A

Amrubicin[20830-81-3]

H H OH H COCH3 NH2 E

These proved the enzymatic mechanism of re-sistance.

Streptomycin is the first aminoglycoside an-tibiotic and the second antibiotic introducedclinically after penicillin, isolated from Strep-tomyces griseus by Waksman [161]. The earlystructural studies have been reviewed [77]. Thetwo anomeric configurations were found to be α-l by application of Hudson’s rules of isorotationand NMR spectral analysis. The absolute struc-ture of streptomycin has been confirmed by X-ray analysis of its oxime selenate [78]. Strepto-mycin has been synthesized by oxidation ofdihydrostreptomycin [79]. Streptomycin showsstrong activity against a wide range of gram-positive and gram-negative bacteria includingMycobacterium. Streptomycin is the first choice

among antituberculotic antibiotics and has beenused for the therapy of Spirochaeta and Tre-ponema infections.

Streptomycin [57-92-1]

20 Antibiotics

Kanamycin was found by Umezawa in theculture broth of Streptomyces kanamyceticusin 1957 [151]. It is produced with other com-ponents, kanamycin B (bekanamycin) and C.Kanamycin shows toxicity much lower than thatof the earlier aminoglycosides, and strong ac-tivity against a wide range of gram-positiveand gram-negative bacteria, including Mycobac-terium. Kanamycin has been used clinically fortreatment of such serious infections as dysen-tery, salmonellosis, and tuberculosis.

Bekanamycin (kanamycin B) is one of thetwo minor components that have been isolatedfrom the culture filtrate of kanamycin-producingStreptomyces kanamyceticus [152]. It shows thesame antibacterial spectrum as kanamycin butwith stronger activity.

Dibekacin (3′,4′-dideoxykanamycin B) wasthe first drug developed on the basis of the enzy-matic mechanism of aminoglycoside resistance.It was synthesized by the removal of the 3′-and 4′-hydroxyl groups of bekanamycin [155].Dibekacin shows excellent activity, as expected,against a variety of bacteria, including kana-mycin-resistant strains. It shows higher activitythan kanamycin against Pseudomonas aerugi-nosa, Proteus, and other pathogens.

Amikacin was synthesized in 1970 by selec-tive N-acylation of kanamycin [156]. Amikacinwas designed based on the mechanisms of bac-terial resistance to kanamycin and related com-pounds in which the 3′-hydroxyl group of theantibiotic is phosphorylated enzymatically. TheN-acyl moiety prevents this enzymatic inactiva-tion.

Tobramycin was found in 1967 in the culturebroth of Streptomyces tenebrarius [153]. Struc-turally it is closely related to kanamycin, i.e.,it is a naturally produced 3′-deoxy derivative ofbekanamycin. The 3′-hydroxyl group was foundto be the target of enzymatic phosphorylationby resistant bacteria. As expected, tobramycinshows strong activity against resistant bacteria,including Pseudomonas aeruginosa, having thisphosphorylating enzyme.

Gentamicin was found in 1963 in the cul-ture broths of Micromonospora purpurea and

M. echinospora as a mixture of at least 16structurally related components [154]. The ma-jor components used in clinical preparationsare gentamicins C1a, C1, and C2. Gentamicinshows high activity against a variety of gram-positive and gram-negative bacteria, includingPseudomonas aeruginosa, Proteus, and Serra-tia. It has been widely used for the clinical treat-ment of serious infections. Gentamicin is usedalone or in combination with β-lactam anti-biotics and is being replaced gradually by thenewer, less toxic aminoglycosides.

Sisomicin was found in the culture broth ofMicromonospora inyoensis in 1970 [164]. Thestructure and activity of sisomicin are very sim-ilar to those of gentamicin C1a, the major com-ponent of the gentamicin complex. Sisomicinshows stronger bacterial activity and lower renaland ototoxicity than gentamicin C1a.

Kanamycin [59-01-8]

Bekanamycin [4696-76-8]

Dibekacin [34493-98-6]

Antibiotics 21

Amikacin [37517-28-5]

Arbekacin [51025-85-5]

Isepamicin [58152-03-7]

Tobramycin [32986-56-4]

Gentamicin C1a [26098-04-4]

Sisomicin [32385-11-8]

Netilmicin [56391-56-1]

Micronomicin [52093-21-7]

Netilmicin was synthesized in 1976 by in-troducing an ethyl group into the 1-amino groupof sisomicin [165]. The molecular design wasbased on the biochemical mechanisms of bacte-rial resistance to the gentamicin–sisomicin an-tibiotic group. The modification at the 1-aminogroup is known to prevent adenylation at the 2′′-hydroxyl group and acetylation at the 3-aminogroup, and to deter acetylation at the 6′-ami-no residue. Netilmicin shows almost the sameactivity against a variety of gram-positive andgram-negative bacteria as sisomicin and strongactivity against gentamicin - sisomicin-resistantbacteria.

Micronomicin was found in the culturebroth of Micromonospora sagamiensis var.nonreducans in 1974 [157]. The methylatedamino group at the 6′-position is not subjectedto the enzymatic acetylation caused by resis-tant bacteria, including Pseudomonas aerugi-

22 Antibiotics

nosa. Micronomicin is less toxic than gentam-icin to the renal and aural systems.

Fradiomycin (Neomycin) is produced byStreptomyces fradiae and by Streptomyces albo-griseolus [159]. It consists of two closely relatedcomponents, fradiomycin B and C, and showsstrong activity against a wide range of gram-positive and gram-negative bacteria, includingSerratia and Pseudomonas aeruginosa. Becauseof its renal and ototoxicity, it is given by oral ortopical application. Because it is not absorbedorally, as are other aminoglycoside antibiotics,it is used orally only for the purpose of sup-pressing intestinal flora, i.e., in treating dysen-tery, salmonellosis, and diarrhea in the pediatricfield. Fradiomycin also has been used topicallyin the treatment of bacterial infections of the eyeand skin.

Ribostamycin was found in the culture brothof Streptomyces ribosidificus in 1970 [158]. Itis structurally related to fradiomycin but lacksthe diaminoidose (glucose) moiety substitutedon the ribose moiety. Ribostamycin is much lesstoxic than fradieomycin and shows strong activ-ity against a variety of gram-positive and gram-negative bacteria except Pseudomonas aerugi-nosa. It is used parenterally for therapy of uri-nary tract, respiratory tract, surgical, and otherinfections.

Paromomycin was found in the culture brothof Streptomyces rimosus forma paromomycinusand of S. Crestomyceticus in 1959 [160].Whereas the antibacterial activity is weakerthan that of fradiomycin, it is much less toxic.Paromomycin is used by intramuscular injectionfor therapy of respiratory, urinary, and surgi-cal infections and by oral administration to treatdysentery and salmonellosis.

Fradiomycin B [119-04-0]

Ribostamycin C [25546-65-0]

Paromomycin I [7542-37-2]

Spectinomycin was found in the culturebroth of Streptomyces spectabilis in 1961 [166].This antibiotic was intended for use in treatingvarious infections, as are the other aminogly-coside antibiotics, but its activity is insufficientfor clinical efficacy except against gonorrhea.Spectinomycin does not develop crossresistancewith any other antibiotic and shows low toxic-ity; it is used by deep intramuscular injectionfor “single-session,” bolus-injection therapy ofgonorrhea.

Spectinomycin [1695-77-8]

Astromicin (fortimicin A) was found in theculture broth of Micromonospora olivoast-erospora in 1976 [167]. It has a unique confor-mation with an acylated diamino inositol moietydifferent from other aminoglycoside antibiotics.Astromicin is produced with one major byprod-uct, fortimicin B, and several minor components.

Antibiotics 23

Astromycin (Fortimicin A) [55779-06-1]

Fortimicin B [55779-06-1]

Kasugamycin was found in the culture brothof Streptomyces kasugaensis by Umezawa in1965 [168]. It has a cyclitol structure and anamidinocarboxylic acid moiety, showing strongactivity against phytopathogenic fungi, espe-cially Pericularia oryzae, the pathogen caus-ing rice blast. Kasugamycin also shows activ-ity against Pseudomonas, and its toxicity is verylow. Kasugamycin has been used in agricultureto protect rice plants against rice blast and foranimal infections.

Kasugamycin [6980-18-3]

Hygromycin B was found in the culturebroth of Streptomyces hygroscopicus in 1953[163]. It showed activity against a variety ofgram-positive and gram-negative bacteria aswell as fungi. Hygromycin B has been used fortherapy of helminthic infections in swine andpoultry.

Destomycin A was found in the culture brothof Streptomyces rimofaciens in 1965 [162]. Itshows activity against a variety of gram-positiveand gram-negative bacteria as well as fungi and,more interestingly, against helminths. Desto-mycin A has been used to treat helminth infec-tions in swine and poultry.

Hygromycin B [31282-04-9]

Destomycin A [14198-35-5]

3.5. Nucleosides

The biological effects associated with metabolicprocesses and specific enzyme control mech-anisms are diverse in naturally occurring nu-cleosides and their synthetic analogs. Nucleo-sides exhibit several biological effects, includ-ing antibiotic, anticancer, and antiviral activi-ties. They possess antimitotic and immunosup-pressive activities and cardiovascular and othereffects [80 – 82]. Moreover, it should be kept inmind that nucleoside analogs can assume otherfunctional roles not as yet recognized, and thatfurther therapeutic applications can be expectedin the future. These analogs are obtained pre-dominantly from microbial sources.

The nucleoside antibiotics consist of a hete-rocyclic base and a sugar or a carbocyclic sugarlinked by a carbon – nitrogen (N-nucleoside) ora carbon – carbon bond (C-nucleoside). The nu-cleoside antibiotics fall somewhat outside thenormal field of antibiotics with respect to theiractivity spectra and hence to their use. They areimportant for use against fungi, viruses, and cer-tain types of cancer cells. Some typical nucleo-side antibiotics are mentioned here.

24 Antibiotics

3.5.1. N-Nucleosides

Bredinin, produced by Eupenicillium brefel-dianum, shows marked immunosuppressive ac-tivity in mice, interferes with replication of Vac-cinia virus in vitro, and inhibits leukemia L 5178cells and Candida albicans [83]. It is used as animmunosuppressive agent for kidney transplan-tation, nephrotic syndrome, lupus nephritis, andrheumatoid arthritis.

Coformycinis isolated from Nocardia inter-forma along with formycin. Coformycin showsa synergistic effect with formycin on Yoshidarat sarcoma cells because of its strong inhibi-tion of adenosine deaminase, which inactivatesformycin. Coformycin, having a characteristicseven-membered-ring base moiety, is thoughtto be a typical example of a “transition-stateanalog” in the adenosine deaminase reaction[84 – 86].

Cordycepin, 3′-deoxyadenosine, was one ofthe first nucleoside antibiotics isolated fromCordyceps militaris. It inhibits Bacillus subtilis,Mycobacterium tuberculosis, KB cell cultures,and Ehrlich ascites tumor cells.

Bredinin [50924-49-7]

Coformycin [11033-22-0]

Cordycepin [73-03-0]

Polyoxins were found in the culture brothof Streptomyces cacaoi var. asoensis in 1965[87 – 89]. It consists of several closely relatedcomponents, polyoxin A to polyoxin O, andshows activity against phytopathogenic fungi byinhibition of cell-wall chitin synthesis. Polyoxinhas been used in agriculture against fungal in-fections, especially Alternaria leaf spot in veg-etables and fruits.

Polyoxin C [21027-33-8] R = OH

Polyoxin I [12886-33-5]

Polyoxin N [37362-29-1] R = OH

Polyoxin O [37362-28-0] R = H

Blasticidin S was found in the culturebroth of Streptomyces griseochromogenes byYonehara in 1958 [97]. It is a pyran-containingnucleoside and shows strong activity againstphytopathogenic fungi, especially Periculariaoryzae, the pathogen causing rice blast. Blas-ticidin S has been used to protect rice plants.

Blasticidin S [2079-00-7]

3.5.2. C-Nucleosides

Formycin is isolated from Nocardia inter-forma and from Streptomyces lavendulae [90,

Antibiotics 25

Name R1 R2 R3

Polyoxin A [19396-03-3] CH2OH OH

Polyoxin B [19396-06-6] CH2OH HO OHPolyoxin D [22976-86-9] COOH HO OHPolyoxin E [22976-87-0] COOH HO HPolyoxin F [23116-76-9] COOH OH

Polyoxin G [22976-88-1] CH2OH HO HPolyoxin H [24695-54-3] CH3 OH

Polyoxin J [22976-89-2] CH3 HO OHPolyoxin K [22886-46-0] H OH

Polyoxin K [22976-90-5] H HO OHPolyoxin M [34718-88-2] H HO H

91]. The antibiotic is effective against Xan-thomonas oryzae and Pellicularia filamentosa.Its activity against Yoshida rat sarcoma cell isenhanced by coformycin. Formycin B inhibitsXanthomonas oryzae and interferes with multi-plication of influenza A virus in the cells of chickchorioallantoic membrane [92].

Showdomycin, isolated from Streptomycesshowdoensis, is very active against Streptococ-cus hemolyticus. It is moderately active againstother gram-positive and gram-negative bacteriaand also effective against Ehrlich ascites tumorin mice and HeLa cells [93].

Formycin [6742-12-7]

Formycin B [13877-76-4]

26 Antibiotics

Showdomycin [16755-07-0]

3.5.3. Carbocyclic Nucleosides

Since the pioneering synthesis of the racemiccarbocyclic analog of adenosine by Shealyand Clayton and the subsequent isolation ofaristeromycin from Streptomyces citricolor, theinterest in this class of compounds has beenrenewed by the isolation of a new carbocyclicnucleoside, neplanocin A. The latter exhibitsremarkable antitumor activity against L 1210leukemia in mice, and its synthetic analogs arenow being studied extensively [94 – 96].

Aristeromycin [19186-33-5]

Neplanocin [72877-50-0]

3.6. Macrolides

Macrolide antibiotics are polyfunctional macro-cyclic lactones and the majority of them containat least one amino sugar, which is the cause ofthe basicity of the molecules. Neutral macrolidescontaining only a neutral sugar moiety are alsoknown. These antibiotics have become targetsin the aldol strategy of organic synthesis toconstruct their polyhydroxy functions stereo-selectively [98, 99]. The antibiotics are clas-sified as either 12-, 14-, or 16-membered ringmacrolides and polyenes.

3.6.1. 12-Membered Ring Macrolides

Methymycin, produced by Streptomycesvenezuelae, was first shown to be a 12-membered lactone, comprising the aglycone ormethynolide and D-desosamine [100, 101].

Methymycin [497-72-3]

3.6.2. 14-Membered Ring Macrolides

Erythromycin, the first of the macrolide anti-biotics, was found in the culture broth of Strep-tomyces erythreus in 1952 [173]. Its chemicalstructure and synthesis have been studied exten-sively by Woodward. Erythromycin shows ac-tivity against gram-positive bacteria and gram-negative cocci as well as Mycoplasma and Lep-tospira. Erythromycin esters are used for oraladministration. An ester, lactobionate, is usedby drip infusion for serious infections. Erythro-mycin derivatives with a modified 14-memberedring, clarithromycin [170] and roxithromycin[171], and ring-expanded derivative, azithro-mycin [172], are also in clinical use.

Erythromycin [114-07-8] R1 = H, R2 = O

Clarithromycin [81103-11-9] R1 = CH3, R2 = O

Roxithromycin [80214-83-1] R1 = H,

R2 = NOCH2OCH2CH2OCH3

Antibiotics 27

Azithromycin [83905-01-5]

3.6.3. 16-Membered Ring Macrolides

Kitasamycin, formerly called leucomycin,was found by Hata in the culture broth of Strep-toverticillium kitasatoensis in 1953 [102]. Thiswas the first macrolide antibiotic with a 16-membered lactone constituent. Kitasamycin isa complex of leucomycin A components and theester forms are used for the treatment of gram-positive bacterial and gram-negative coccal in-fections, as well as infections of Mycoplasma,Spirochaeta, and Treponema by injection or byoral topical administration.

Josamycin was found in the culture broth ofStreptomyces narboensis var. josamyceticus in1964 [174]. Its identity with leucomycin A3 wasconfirmed by structural study.

Midecamycin was found in the culture brothof Streptomyces mycarofaciens in 1971 [176].Under specific culture conditions, it is producedby the organism as a single component. Mideca-mycin shows almost the same antimicrobialspectrum and activity as those of kitasamycin.Although its serum and urine concentrations arelow, it distributes in tissues at high concentra-tion after the oral administration. Rokitamycinis a propanoyl derivative of leukomycin.

Leucomycin A1 [16846-34-7] R1 = H,R2 = COCH2CH(CH3)2, R3 = HLeucomycin A3 (Josamycin) [16846-24-5] R1 = COCH3,R2 = COCH2CH(CH3)2, R3 = HLeucomycin A4 [18361-46-1] R1 = COCH3,R2 = COCH2CH2CH3, R3 = HLeucomycin A5 [18361-45-0] R1 = H,R2 = COCH2CH2CH3, R3 = HLeucomycin A6 [18361-48-3] R1 = COCH3,R2 = COCH2CH3, R3 = HLeucomycin A7 [18361-47-2] R1 = H, R2 = COCH2 CH3,R3 = HLeucomycin A8 [18361-50-7] R1 = COCH3, R2 =COCH3, R3 = HLeucomycin A9 [18361-49-4] R1 = H, R2 = COCH3,R3 = HLeucomycin A13 [78897-52-6] R1 = H,R2 = CO(CH2)4CH3, R3 = HMidecamycin [34547-80-8] R1 = COCH2CH3,R2 = COCH2CH3, R3 = HRokitamycin [74014-51-0] R1 = H, R2 = COCH2CH3,

R3 = COCH2CH3

Spiramycins were found in the culture brothof Streptomyces ambofaciens in 1954 [103] andtheir acetates were synthesized in 1965. Spira-mycin shows almost the same antimicrobialspectrum and activity as the other macrolides,but its acetate, acetylspiramycin, has much bet-ter pharmacokinetic properties and activity invivo.

Acetylspiramycin II [87111-42-0] R = COCH3

Acetylspiramycin III [112501-15-2] R = COCH2CH3

Tylosin was found in the culture broth ofStreptomyces fradiae in 1959 [104 – 106]. Itsantimicrobial spectrum is the same as that ofthe other macrolide antibiotics, and its activityagainst Mycoplasma is the widest and highest ofany member of its group. Against gram-positivebacteria it is slightly weaker than erythromycin.

28 Antibiotics

Tylosin shows a very high and long-term tis-sue concentration when administered subcuta-neously. It is used by injection or oral adminis-tration to treat Mycoplasma and gram-positivebacterial infections in poultry, swine, and otherlivestock.

Ivermectin was synthesized starting withavermectin, which was found in the culturebroth of Streptomyces avermitilis in 1977 [175].Unlike other antibiotics, its activity is strictlyagainst insects, mites, and animal parasites. Iver-mectin has been used against pests, mites, andother parasites in domestic animals and live-stock.

Tylosin [1401-69-0]

Ivermectin B1a [70161-11-4] R = CH2CH3

Ivermectin B1b [70209-81-3] R = CH3

3.6.4. Polyenes

Amphotericin B was found in the myceliumof Streptomyces nodosus M-4575 by Gold in1956 [183]. It is produced with another polyenemacrolide antibiotic, amphotericin A, and sep-arated by solvent extraction. Amphotericin Bshows strong antimycotic activity against a va-riety of fungi and pathogenic yeasts (Candida)and is used by injection and as a vaginal suppos-itory.

Nystatin was found in the mycelium ofStreptomyces noursei in 1950 [184]. Nystatinwas used orally and topically as the first clini-cally applied polyene macrolide with antifungalproperties. Nystatin shows activity against Can-dida and filamentous fungi and is used to treatCandida infections of the mouth, digestive or-gans, and vagina. The application of nystatin incombination with gentamicin and vancomycinto sterilize the gut in perioperation of bone-marrow transplantation has been developed.

Amphotericin B [1397-89-3]

Nystatin A1 [34786-70-4]

3.7. Ansamycins

The ansamycins have an aliphatic “ansa” bridgethat connects two nonadjacent positions of anaromatic system [107].

The rifamycins are produced by Nocardiamediterranei [108, 109]. Rifamycin B, rifa-mycin O, and rifamycin S were found in thefermentation products. Rifamycin B was mod-erately effective against gram-positive bacteria.The oxidation of rifamycin B gave rifamycinO, which can be hydrolyzed to the more ac-tive rifamycin S. The latter can be reduced torifamycin SV with ascorbic acid. RifamycinSV is converted to the therapeutically impor-tant rifampicin. Rifampicin has strong biolog-

Antibiotics 29

ical activity against gram-positive microorgan-isms and mycobacteria, particularly Mycobac-terium tuberculosis and Mycobacterium leprae[110 – 112].

Rifamycin B [13929-35-6]

Rifamycin O [14487-05-9]

Rifamycin S [13553-79-2]

Rifamycin SV [6998-60-3]

Rifampicin [13292-46-1]

3.8. Peptides

Various low molecular mass peptides, oligopep-tides, and protein-like substances are foundamong the antibiotics of microbial origin. Pep-tide antibiotics differ from the proteins and pep-tides of higher animals and plants in many re-spects [113]. The following characteristics fre-quently are found in the peptide antibiotics:1) Molecular masses of the peptide antibiotics

are smaller (in the range of 500 – 1500) thanthose of peptide hormones.

2) Peptide antibiotics contain some uncommonamino acids that are not found in proteins andpeptide hormones of animal or plant origin.

3) Lipids and other non-amino acid structuresare found in many peptide antibiotics.

4) Peptide antibiotics frequently contain d-ami-no acids.

5) Virtually all of the peptide antibiotics resisthydrolysis by proteolytic enzymes.

6) The antibiotics are often cyclic peptides.7) Families of closely related peptide antibiotics

are frequently produced by the same microor-ganism.Uncommon amino acid constituents of

peptide antibiotics are: Dab = α,γ-diamino-butyric acid; Orn = ornithine; MeVal = N-methylvaline; MeGly = N-methylglycine; Sar =sarcosine; MOA = 6-methyloctanoic acid; IOA= isooctanoic acid. The arrow indicates the di-rection of the amide bond between the aminoacids. Thus, the arrow begins where the car-bonyl group attaches and ends where the aminogroup attaches (− CO − NH→).

Gramicidin S was found in the culture brothof Bacillus brevis [118]. It is a basic peptideshowing strong activity against gram-positive

30 Antibiotics

bacteria and considerable activity against gram-negative cocci and Mycobacterium. GramicidinS is rather toxic but is not orally absorbed; itis used topically as an ointment or as eye orear drops in combination with other antibacterialdrugs.

Gramicidin S [113-73-5]

Polymyxin B was isolated from Bacilluspolymyxa [177]. It was later separated into themajor component polymyxin B1 and the mi-nor component polymyxin B2. Polymyxin B isa basic polypeptide and shows strong activityagainst gram-negative bacteria, but its activityagainst gram-positive bacteria, Mycobacterium,and fungi is weak. Because of its toxicity, itis used carefully by intramuscular injection forresistant Pseudomonas aeruginosa infections,e.g., sepsis. Polymyxin B is used orally to ster-ilize the gut in leukemic patients, intraspinallyfor meningitis, or topically.

Colistin was found in the culture broth ofBacillus polymyxa var. colistinus in 1950 [179].It is closely related to polymyxin and showsstrong activity against gram-negative bacteriaincluding Pseudomonas aeruginosa. Colistinand its methanesulfonic acid derivative havebeen used to treat urinary tract infections causedby Escherichia coli and P. aeruginosa. Theyhave also been used parenterally to treat dysen-tery and abdominal infections and topically forophthalmic and otorhinolaryngological infec-tions.

R X Y ZPolymyxin B1[4135-11-9]

MOA Phe Leu l-Dad

Polymyxin B2[34503-87-2]

IOA Phe Leu l-Dad

Colistin A[7722-44-3]

MOA Phe Leu l-Dad

Colistin B[7239-48-7]

IOA Leu Leu l-Dad

Bacitracin was found as a polypeptide com-plex in the culture broth of Bacillus subtilis andB. licheniformis in 1945 [178]. It was first used asa mixture of at least nine bacitracin components.The structure of bacitracin A was determined in1966 [119].

Bacitracin A [1402-99-9]

Vancomycin was found in the culture brothof Streptomyces orientalis [180]. It is a large mo-lecular glycopeptide showing bactericidal activ-ity against gram-positive bacteria and anaerobes.It is used to treat resistant infections of Staphylo-coccus, to sterilize the gut in the perioperation ofbone-marrow transplantation, and in leukemicpatients. Recently its efficacy has been demon-strated against pseudomembrane colitis, whichis caused by Clostridium difficile. Teicoplanin isused for the treatment of aerobic and anaerobicgram-positive bacteria [181].

Vancomycin [1404-90-6]

Antibiotics 31

Teicoplanin A2−1 [91032-34-7]

R3 =

Teicoplanin A2−2 [91032-26-7]

R3 =

Teicoplanin A2−3 [91032-36-9]

R3 =

Teicoplanin A2−4 [91032-37-0]

R3 =

Teicoplanin A2−5 [91032-38-1]

R3 =

Teicoplanin A3−1 [93616-27-4]

R2 =H

Actinomycins (mixtures of A, B, C, andother components) were first found by Waksmanin 1940 in the culture broth of Streptomyces an-tibioticus [182]. Actinomycin D was found bythe same group in 1954, obtained as a singlecomponent from S. parvullus. Its strong activ-ity against Wilm’s tumor and other cancers hasbeen evaluated.

Actinomycin D [50-76-0]

Bleomycins constitute a group of glycopep-tide antibiotics containing unusual amino acidresidues and a disaccharide of uncommon sug-ars produced by Streptomyces verticillus, effec-tive against squamous cell carcinoma and malig-nant lymphoma [114]. The complete structurehas been elucidated by chemical studies and X-ray crystallographic analysis of a biosyntheticintermediate [115, 116]. This structure has beenverified by total synthesis. The naturally occur-ring bleomycins are obtained in copper-chelatedform as a mixture of congeners that differ onlyin the C terminus substituents. Metal-free bleo-mycin can be prepared by treatment with hydro-gen sulfide. A mixture of metal-free bleomycinA2 (55 – 70 %) and B2 (25 – 32 %) has beenused for clinical treatment because the mixturehas an effect superior to that of A2 alone on hu-man squamous cell carcinoma. The copper ion inbleomycin is replaced by iron after administra-tion to form a bleomycin–iron complex that ex-erts antitumor activity. More than 300 bleomycinanalogs have been prepared by chemical modifi-cations or fermentations. Pepleomycin possess-ing improved properties has been brought intoclinical use [117].

32 Antibiotics

Bleomycin A2 [11116-31-7]

Bleomycin B2 [9060-11-1]

Pepleomycin [68247-85-8]

3.9. Enediynes

A class of antitumor antibiotics possessing char-acteristic enediyne chromophores have beenisolated [131]. These include neocarzinostatin[132, 133], calicheamicins [134, 135], esperam-icins [136, 137], dynemicin A [138].

Neocarzinostatin was isolated from Strepto-myces carzinostaticus Var. F-41 by Ishida in1957 as a complex of a chromophore and anapoprotein [132, 133]. It showed strong cytotox-icity against sarcoma 180 ascites, tumor cells,and leukemia SN-36. A polymer-conjugatedderivative of neocarzinostatin was prepared andadministered via the tumor-feeding artery show-ing increased stability in blood and the immuno-genicity was much lower than the parental neo-carzinostatin [142]. The role of the apoprotein isto stabilize the enediyne chromophore that hasa 9-membered ring with a double bond and twotriple bonds.

Ala – Ala – Pro – Thr – Ala – Thr – Val – Thr – Pro – Ser

– Ser –

Gly – Leu – Ser – Asp – Gly – Thr – Val – Val – Lys – Val

– Ala –

Gly – Ala – Gly – Leu – Gln – Ala – Gly – Thr – Ala –

Tyr – Asp –

Val – Gly – Gln – Cys – Ala – Ser – Val – Asn – Thr – Gly

– Val –

Leu – Trp – Asn – Ser – Val – Thr – Ala – Ala – Gly – Ser

– Ala –

Cys – Asx – Pro – Ala – Asn – Phe – Ser – Leu – Thr –

Val – Arg –

Arg – Ser – Phe – Glu – Gly – Phe – Leu – Phe – Asp –

Gly – Thr –

Arg – Trp – Gly – Thr – Val – Asx – Cys – Thr – Thr –

Ala – Ala –

Cys – Gln – Val – Gly – Leu – Ser – Asp – Ala – Ala –

Gly – Asp –

Gly – Glu – Pro – Gly – Val – Ala – Ile – Ser – Phe – Asn

Neocarzinostatin [9014-02-2]

The anticancer activity of neocarzinostatin isdue to its capability to cleave DNA. The DNAdamage is initiated by nucleophilic attack atC12 of neocarzinostatin chromophore by a sul-fur nucleophile, which leads to the formationof a labile cumulene intermediate that under-goes a facile cycloaromatization [139]. The re-sulting biradical abstracts hydrogen atom of thedeoxyribose of DNA (see top of page 33).

Similar biradical formation via the Bergmancyclization of the other enediyne class anti-biotics, calicheamicin γI

1, esperamicin A1, anddynemicin A, has been proposed [131]. My-lotarg, a CD33 antibody linked to calicheamicin,is useful in targeting therapy for acute myeloidleukemia [140, 141].

Calicheamicin γ′1 [108212-75-5]

Antibiotics 33

Esperamicin A1 [99674-26-7]

Dynemicin A [124412-57-3]

3.10. Other Important Antibiotics

Cycloserine, d-4-amino-3-isoxazolidine,was isolated from many Streptomyces species[120]. Cycloserine is now produced solely bychemical synthesis and used particularly fortuberculosis of the lungs and for leprosy withp-aminosalicylic acid or isonicotinic acid hy-drazide.

Cycloserine [68-41-7]

Griseofulvin is produced by Penicilliumgriseofulvum, P. janczewskii, and Nigrosporaoryzae. It is unique in possessing the spiro-carbon moiety [121, 122]. Griseofulvin is veryactive against fungi, and it is used orally to treatfungal infections of human skin.

34 Antibiotics

Griseofulvin [126-07-8]

Chloramphenicol, the first of the so-calledbroad-spectrum medicinal antibiotics, was orig-inally obtained from Streptomyces venezuelaein 1947 [123]. It is now manufactured by achemical process. Chloramphenicol is activeagainst rickettsia, chlamydiae, and mycoplas-mas, as well as a wide range of gram-positive andgram-negative bacteria. However, use is limitedby the risk of bone marrow damage or aplasticanemia at too high or too prolonged application[124].

Chloramphenicol [56-75-7]

Mitomycins have unique chemical struc-tures in which three different carcinostatic func-tions – aziridine, carbamate, and quinone – arearranged around a pyrro[1,2-a]indole nucleus[125]. The first mitomycins were discoveredin 1956 by Hata in a culture filtrate of Strep-tomyces caespitosus. These compounds, desig-nated mitomycins A and B, show highly potentantibacterial activity and moderate antitumor ac-tivity, but they are quite toxic in mice. In 1958,mitomycin C, an extremely valuable antitumordrug, was isolated from Streptomyces caespi-tosus [126, 127]. In 1960, another mitomycin,porfiromycin, was isolated from Streptomycesardus.

Name X Y ZMitomycin A [4055-39-4] OCH3 OCH3 HMitomycin B [4055-40-7] OCH3 OH CH3

Mitomycin C [50-07-7] NH2 OCH3 HPorfiromycin [801-52-5] NH2 OCH3 CH3

Fosfomycin was found in the culture brothof Streptomyces fradiae in 1967 [128]. Itschemical structure is simple and unique amongantibiotics in having a C−P bond. Fos-fomycin shows antibacterial activity againstgram-positive and gram-negative organisms, in-cluding Pseudomonas aeruginosa and Serra-tia marcescens, and β-lactam-resistant Staphy-lococcus aureus. It shows no cross-resistancewith other classes of antibiotics.

Fosfomycin [23155-02-4]

Fusidic acid was found in the culture brothof a fungus imperfectus, Fusidium coccineum, in1962 [129]. It has a steroidal structure but showsno hormonal activity. Fusidic acid shows verystrong activity against Staphylococcus aureusand weak activity against other gram-positivebacteria and gram-negative cocci and Mycobac-terium. Its clinical use is restricted to staphy-lococcal infections resistant to other classes ofantibiotics.

Fusidic acid [6990-06-3]

4. Antibiotic Resistance

An organism becomes resistant to an antibiotic ifit survives continued contact. Antibiotics repressthe growth of the sensitive organism in a culture,which results in the survival of naturally resis-tant organisms. Microbial resistance can be ac-quired through a spontaneous or induced muta-tion. Various examples of cross-resistance havebeen observed and several cross-resistant groupsof antibiotics are recognized. Combined use of

Antibiotics 35

two antibiotics retards the appearance of resis-tant organisms.

Microorganisms develop antibiotic resis-tance by several mechanisms.

• Microorganisms inactivate or modify anti-biotics. β-Lactam antibiotics are inactivatedthrough the production of β-lactamases,such as penicillinase or cephalosporinase.Aminoglycosides are inactivated by O-phosphorylation, O-nucleotidylation, and N-acetylation. Semisynthetic β-lactams andaminoglycosides have been developed toovercome the resistance.

• Microorganisms alter the proteins that aretarget of the antibiotics. Methicillin-resistantStaphyllococcus aureus (MRSA) was first re-ported in 1961 in UK and spreaded widelyin hospitals. MRSA acquired a gene that en-codes an altered penicillin-binding protein towhichβ-lactam antibiotics bind with reducedaffinity [130]. Glycopeptide antibiotics van-comycin and teicoplanin are used for thetreatment of MRSA.

5. Fermentation

Fermentation is considered here from the fol-lowing points of view:

1) Biological development, which includesscreening and selection, mutation, and main-tenance of the strain.

2) Process development leading to large-scalemanufacture.

3) Improvements in fermentation technology.

5.1. Screening

Technical developments in the production ofpenicillin have given the field new momentumand have stimulated the search, not only for moreefficient strains, but also for microorganismsthat produce completely different antibiotics.This process is called screening because valu-able antibiotic producers are separated from thelarge number of organisms found in nature. Thescreening process and the expected results areinfluenced by several factors.

Source of Sample. Worldwide screeningendeavors to isolate the individual microorgan-isms not only from soil samples from differentsources, but also from other microbe-containingmaterials. Samples from unusual sources oftenshow the occurrence of selection and adaptation.For example, thermophilic microorganisms areexamined in samples taken from deep caves, thesea bottom, hot springs, or geysers.

Examination Technique. There are severalfactors that determine the conditions underwhich a certain microorganism not only livesand grows but also efficiently produces its an-tibiotic. These factors include the compositionand pH of the culture medium, the additives, theair supply, and the temperature. These factorsare also of prime importance for any later in-dustrial fermentation. The isolation and testingof the new antibiotic, first in vitro and then inanimal experiments, and the indisputable proofthat the new compound is not identical to one ofthe numerous known antibiotics are part of theexamination technique.

Purpose of the Examination. In the earlydays of antibiotic screening, any organism thatshowed antibiotic activity was screened, but laterdefinite objectives were set and appropriate ex-amination techniques were developed. The fac-tors mentioned in the previous paragraph narrowthe choice to certain bacteria and fungi. Furtherrestrictions are brought about by the selection oforganisms used to test the efficacy of the antibi-otic. Such specific screening methods are usedto find, e.g., antifungal agents, antibiotics activeagainst cancer or viruses, or antibiotics effectiveagainst bacteria resistant to other antibiotics.

A general overview of the successes andfailures experienced during the search for anti-biotics has been presented [185]. Goulden [186]reported that in the United States from 1955to 1966, about 90 000 synthetic compounds, 20000 plant extracts, and 120 000 culture solutionsof microorganisms were tested against differ-ent types of neoplasms. About 1600 substancesshowed sufficient activity to justify their pu-rification; 31 fermentation products reached thefirst clinical test, but only five of them got as faras the second step. Of these only two products,mithramycin and streptonigrin, are clinicallyused today. It can be concluded that, starting with

36 Antibiotics

a limited number of samples, the probability ofobtaining a therapeutic agent is extremely low.This also applies to other screening objectives,e.g., the search for antibiotics more effectiveagainst tuberculosis, resistant microbes, or fungiand yeasts. In order to realize a definite goal, newtest methods had to be introduced. Asteromycinwas discovered in the process of introducing newtests against mycoplasmas. A search for anti-biotics active against bacteriophages led to thediscovery of a strain producing dextrochrysin.Dienomycin was found when testing nucleotideswith Wood’s reagent. The leucopeptines, whichare active against phytopathogenic microorgan-isms, gram-positive bacteria, and mycobacteria,were discovered as a result of their antiplasminactivity. Although all these antibiotics have notbeen approved for use, they show the importanceof new screening methods.

5.2. Selection, Mutation, andMaintenance of Strains

The biological production of antibiotics is car-ried out predominantly by microorganisms. Thediscovery and isolation of the microorganismare the first steps of a long process leadingto the production of the antibiotic. A yield-improvment program, a very time-consumingprocess, is needed to raise the yield of the strainto an economic level [187]. This is done pri-marily by developing optimal cultivation con-ditions, keeping in mind that the deep-tank andsubmerged methods are the only ones techni-cally applicable. Even so, the concentration ofantibiotic in the culture medium is generally notenough to start production.

For this reason selection is necessary. A largenumber of single individuals belonging to astrain are isolated. These are bred, and the antibi-otic production in the cultures is quantitativelymeasured. New individuals with good, average,poor, or even no productivity usually develop.Hence, selection is carried out from generationto generation in an effort to develop a strainwith as high an antibiotic productivity as pos-sible, one that produces few interfering byprod-ucts (dyes, toxins, other antibiotics, etc.), andone that remains stable over a long period oftime, i.e., one whose antibiotic production doesnot decline.

Another technique used to obtain improvedstrains is mutation. Cultures are exposed system-atically to mutagens, such as ultraviolet radiationor specific chemical compounds. The dosage ischosen so that of a very large number of treatedindividuals only a few survive, and these are ge-netically altered. The mutants obtained in thisway are generally valueless. In a few cases, how-ever, it is possible to separate a single organismthat possesses properties, such as increased an-tibiotic production and strain stability, superiorto those of the untreated strain.

Penicillin production has been perfected tosuch an extent that today’s industrial strains pro-duce at least 35000 U/mL of culture medium. Ona smaller scale, still higher production rates havebeen obtained. The maintenance of the strain,i.e., the production, choice, testing, and storageof efficient antibiotic producers, plays a very im-portant part in the manufacture of antibiotics.The yield of antibiotic tends to decrease throughmany successive rounds of selection. This ten-dency must be monitored using tests in cultureplates, which include methods using the agar dif-fusion test, photocytometry, and tests in shakenerlenmeyer flasks or in small fermenters withvolumes ranging from 10 to 3000 L.

5.3. Process Development Leading toLarge-Scale Production

After an efficient microorganism has been foundin the laboratory, the strain must be broughtinto large-scale production. This process, knownas scaling up, is undertaken in steps and posestremendous technical problems. Several factorsare important in scaling up.

Transition to Larger Volumes. Microbialgrowth, begun in the laboratory on culture plates,transferred subsequently to shaking flasks hav-ing a maximum volume of one liter, and laterto small industrial fermenters having a workingvolume of 3000 L, must ultimately be carried outin production fermenters having a total capacityof about 150 m3 or more. The main probleminvolved in this process of scaling up is to mod-ify the fermentation conditions in such a waythat the same yields are obtained in the largerfermenters as in the smaller ones.

Antibiotics 37

Changes in the Fermenter Geometry. Thestructure of the fermenter, its construction, andits dimensions, greatly affect the yield obtained.The height versus the diameter of the fermenter,the stirring and aeration systems, the coolingsystem (jacket, spiral, or inserted cooling), andthe protection of the inlets and outlets of the fer-menter against infection (pressure sealing) areall important factors in the fermentation process.

Experiments in large-scale fermenters aretime consuming and expensive. Critical com-parisons of similar experiments conducted bydifferent institutes or companies must take intoaccount the fact that the research laboratorieshave only a few types of fermenters at their dis-posal. In addition, almost every researcher han-dles similar fermentation problems using differ-ent strains of a microorganism. Hence, a realcomparison cannot be made and only reservedconclusions can be drawn.

Variations in the Culture Medium or Nu-trient Solution. The culture medium influencesthe growth of the microorganism and, inde-pendently of this, the amount of antibiotic itproduces. The growth media or nutrient solu-tions required for the prefermentation treatment,which primarily must support the rapid multipli-cation of the microorganisms as a monoculture,have a different composition than the nutrientsolution used during fermentation. For example,in fermentation the carbon source should not betoo plentiful. As a result of rapid consumption,nutrients must be resupplied to prevent a nutrientshortage.

In the choice and supply of nutrients, factorsother than the achievement of an optimal antibi-otic production must be considered. A nutrientsuitable for improving the yield of an antibioticmay simultaneously hinder its recovery. Only anaccurate comparison of the yield with the effortrequired, from the prefermentation treatment tothe final product, can decide whether an appar-ently good fermentation raw material is also suit-able for production. The addition or removal ofcertain substances has a direct effect on the an-tibiotic production. In the manufacture of peni-cillin, the addition of building blocks or precur-sors to the fermentation broth causes, dependingon the addition, a preferred production of ben-zylpenicillin (addition of phenylacetic acid) orof phenoxymethylpenicillin (addition of phen-

oxyacetic acid). On the other hand, if chlo-ride ions are largely removed from the culturemedium, e.g., by pretreatment with silver salts[188, 189], or by ion exchange [190], the pro-duction of chlortetracycline is suppressed in fa-vor of tetracycline. Certain inhibitors, e.g., inor-ganic additives, such as bromides and thiocya-nates [191], and a great number of organic com-pounds, also suppress the production of chlor-tetracycline.

Variation of Other Fermentation Condi-tions. Strict control must be maintained duringfermentation. The temperature, the pH (includ-ing the effects of nutrients and additives on thepH), the stirrer speed, the air supply, and the du-ration of fermentation must be monitored con-stantly.

Control of Fermentation by Means of Ad-ditives. A resting surface culture or a sim-ple shaken culture contains a definite nutri-ent medium, which is required to support thegrowth of the microorganism. The growing cul-ture eventually slows down and ceases growth,usually because the medium is spent. A sub-merged fermenter allows the sterile addition ofadditives during the course of fermentation. Inthis way important changes can be made. Asterile air supply and its generally continuousdistribution are vital to all aerobic microorgan-isms. Any interruption of the air supply must beavoided and the air must be evenly distributedthroughout the fermenter. This is achieved by amixing and air-distributing system. The air sup-ply is often limited in large fermenters as a resultof high viscosity and foaming, which makes theaddition of antifoam substances necessary (oils,silicones). Mechanical foam destroyers can alsobe used but they consume large amounts of en-ergy and are not applicable in production plants.The addition of nutrients, in portions or con-tinuously, permits the supply of nutrients to beadjusted at each stage in the fermentation pro-cess. The added nutrients may be organic (e.g.,sugar as the carbon source) or inorganic (e.g.,ammonia as the nitrogen source).

The pH can be adjusted during fermentationby the addition of acid or base. However, expe-rience has shown that the addition of certain nu-trients causes a simultaneous change in the pH.Slower adjustment is physiologically preferable

38 Antibiotics

in this case. The addition of sugar often causes afall in pH because of carbon dioxide formation;peptides and amino acids cause an increase inpH (via the formation of ammonia or other ni-trogen bases). The addition of building blocks,precursors, and inhibitors during the course offermentation has proved useful, especially forthe production of penicillins and tetracyclines.In such cases, a single addition at the beginningof the fermentation procedure leads to a con-centration toxic to the fungus, but because of itsrapid consumption, the precursor concentrationshould be maintained at a certain level.

Measurement and Control Techniques;Analytical Measurements during Fermenta-tion. The process of fermentation is relativelylong, and the antibiotic production is very sen-sitive to disturbances. Precise analytical mea-surements and rapid and accurate control mech-anisms are therefore required.

The monitoring of conditions during thecourse of fermentation can be divided into directmeasurements in or at the fermenter and indirecttesting in the laboratory of samples withdrawnat regular intervals and under sterile conditions.

The direct measurements are immediate andcan sometimes be automated. For example, anelectrode could be installed to monitor the foamlevel and automatically release an antifoam ad-ditive as required. The formation of foam is in-fluenced, within certain limits, by changes in theair supply. Equipment to monitor and control thetemperature (by adjusting the amount of coolingwater) and the pH of the medium is common.Some other direct measurements are the deter-mination of the weight of the full fermenter, e.g.,with the help of a pressure gage (especially atthe start of fermentation and at harvesting), themeasurement and control of the stirring speedand of the air supply, and the determination ofthe partial pressures of oxygen and carbon diox-ide in the fermenter and their concentrations inthe exhaust gas.

Computer monitoring is of great help in in-dustrial production because of the large numberand size of the fermenters. The results of mea-surements on indirect samples, which are avail-able after hours or a few days, also are fed intothe computer.

The tendency today is to analyze numerousfermentation samples, taken at as short intervals

as possible, using automatic analyzing instru-ments for sugar, nitrogen, phosphates, biomass,product, etc. Methods that permit the fully au-tomatic withdrawal of samples during fermen-tation and the automatic transfer of the samplesto different analyzers have been perfected.

Scale Down. When a fermentation proce-dure is carried out for the first time on an in-dustrial scale, scale-up problems occur. Aftertheir start-up problems have been overcome,large fermenters often produce disproportion-ately larger amounts than the previously usedsmaller fermenters. It is difficult to explain thisphenomenon when the same strain and approx-imately the same fermentation conditions areused. This leads to the scale-down problem, i.e.,the problem of increasing the yield obtainedfrom the smaller fermenter to that of the larger.

Solution of the scale-down problem is veryimportant for the further development of a strain.A new, promising mutant or variant developedduring a large-scale fermentation must first beevaluated in a small fermenter. The yield thusobtained must be comparable to the yield ob-tained using the original production strain, alsoin a small fermenter.

Continuous Fermentation. Continuous fer-mentation has proved to be feasible in breed-ing yeast, in the activated-sludge purification ofwaste water, and in the brewing of beer [192,196]. Continuous industrial production of anti-biotics suffers from several difficulties, and it hasmade little progress in displacing batch methods.

1) It is very difficult to keep the yield constant.The highly efficient strains used today tend todegenerate; i.e., the antibiotic production de-clines. This process makes the maintenanceof the strain very important.

2) Maintaining the sterility of the fermentationenvironment and the additives is much moredifficult than in a batch process.

3) A purely technical problem is the continuousaccumulation of culture solutions; this gener-ally necessitates continuous further process-ing. It therefore becomes necessary to con-vert a factory normally working only daysinto one working round-the-clock shifts.

4) The main saving in introducing a continuousprocess lies in the reduction of the volume of

Antibiotics 39

the equipment. However, the extent of spacesaving is directly proportional to the rate ofthe reaction. When the time required for fer-mentation is two weeks or less, then the vol-ume of equipment required for a continuousprocess is scarcely less than that needed fora batch process. In addition, the constructionof the equipment required for a continuousprocess is more complicated and more ex-pensive.Even in the case of antibiotics produced by

fast-growing bacteria, e.g., tyrothricin [197] andgramicidin S [198], reasons 1, 2, and 3, alongwith higher equipment and factory costs, speakagainst continuous production.

5.4. Fermentation Technology

The essential prerequisites for the production ofantibiotics using either submerged fermentationor other fermentation methods are the same.1) A strain of microorganism should produce

the desired antibiotic in satisfactory amountsand, as far as possible, without unwanted by-products that are difficult to remove. It strainshould be as stable as possible; i.e., the pro-duction of antibiotic should not decrease withtime. The strain should also be resistant toother microorganisms, phages, etc.

2) Complete industrial facilities, which includelaboratories for the preparation of inoculumand for the maintenance of the strain and ves-sels for the prefermentation treatment and forfermentation, must be available. The vesselsmust be equipped with devices such as tem-perature regulators, automatic foam destroy-ers, and appliances for the addition of nutri-ents and for the supply of sterile air. In ad-dition, a sufficiently large recovery plant andenough storage space for raw materials, fer-mentation aids, intermediates, and finishedproducts must be available.

3) The fermentation process and its optimaloperation, the properties of the antibioticformed, its isolation, and its efficient purifi-cation must be known in detail.

4) Analytical equipment and methods to moni-tor the operation of the fermentation and re-covery processes and to control the raw ma-terials, intermediates, and end products mustbe available.

Figure 1 shows a schematic outline of thelarge-scale production of penicillin, an exampleof a fermentation process.

5.4.1. Maintenance of the Strain andProduction of Inoculum

The strain of microorganism is maintained as apure culture in a microbiological laboratory. Theunderlying principle is preservation; i.e., a formof the microorganism that is as stable as possiblemust always be available. The microorganism isstored in a large number of small, separate am-pules or vials that are used successively. Culturesof good colonies are constantly restarted so thatthe strain is never depleted.

If the microorganism forms spores, its storageis relatively easy. The spores, a resting form, aredried, usually mixed with sterile soil, and storedin ampules. Spores can be stored for months oryears.

The application of frozen inoculum, stored inthe vegetative state, has advantages. This formis easy to prepare in large amounts and germina-tion is no longer necessary. However, the storagetimes are limited.

The inoculum for penicillin fermentation isproduced by placing the spore-containing soilin a sterile agar nutrient medium in Roux bot-tles and incubating at 24 ◦C. The spores ger-minate in one or two days (vegetative form). Arich mycelium network is formed, from whichnew spores develop in a few days. These young,freshly formed spores are removed from the fun-gal network under sterile conditions and withwater or normal saline. They are then transferredto erlenmeyer flasks containing a suitable ster-ile nutrient solution. The suspension of sporesis shaken at 24 ◦C, enabling them to undergomultiplication.

The inoculum is transferred to another shakenflask and is allowed to grow in nutrient solutionuntil a submerged culture can be started. Thenext steps (Fig. 1) lead to rapid growth and anincrease in volume until finally enough mass ofmycelium is obtained to inoculate the produc-tion fermenter.

Besides breeding the inoculation material,the microbiology laboratory has the equally im-portant task of guaranteeing the maintenance

40 Antibiotics

Figure 1. Schematic outline of the manufacture of penicillina) Antifoam substance; b) Steam; c) Precursors for penicillin formation; d) Condensate; e) Air; f) Air filter; g) Air-flowrecorder; h) Cooling brine; i) Cooling water; k) Spore culture (filled into l); l) Fungal culture with spores; m) Spore sus-pension; n) Prefermenter (inoculation culture for o); o) Intermediate fermenter (inoculation culture for p); p) Productionfermenter; q) Cooling tank; r) Filtration unit; s) Filtrate container; t) Starting vessel for nutrient solution; u) Pump.

and care of the strain, which insures a steadysupply of a microorganism with a constant effi-ciency. If the laboratory limited itself to breed-ing, storage, and regular reinoculation, the an-tibiotic activity of the fermentation cultureswould very soon decrease because these highlyproductive strains tend to mutate and degenerate.To avoid a decrease in the antibiotic productionin industrial fermentation, efficient strains mustbe subjected annually to several thousand singleselections, and the resultant colonies must betested. The single strains thereby isolated showconsiderable differences in their stability, i.e., intheir tendency to develop into good or bad pro-ducers or even into nonproducers. Only after theminimum number of selections necessary for themaintenance of a strain has been exceeded, dothe chances of surpassing the efficiency of theoriginal strain increase. Then an improvementin the factory productivity becomes possible.

5.4.2. Treatment Before and DuringFermentation

The manufacture of antibiotics by means of fer-mentation is always carried out in closed, sterilevessels constructed of stainless steel or of steellined with stainless steel. Figure 2 shows a typ-ical construction.

The supply of sterile air for fermentation isvery important. Foreign organisms are filteredfrom the air by means of glass wool, a filteringcandle, or other methods.

The composition of the nutrient solutionsmust meet the nutritional needs of the microor-ganism; these needs vary depending on the stageof fermentation. The solutions are produced inseparate vessels and are sterilized therein, in thefermenter itself, or in a continuous-flow heat ex-changer. This heater is also used to sterilize theadditives [195].

Antibiotics 41

Figure 2. Schematic outline of a fermenter

The amounts of raw material required mayonly be transported and stored in silos. Hy-draulic transport and weighing with a pressuregage have replaced conventional methods, and

only minor additives, e.g., trace element salts,are weighed in the normal way.

Balance Studies. The balance of energy andmaterials in the particular case of benzylpeni-cillin has been described [199] The manufac-ture of 100 kg of the sodium salt of penicillinrequires 1.2 t carbohydrates, 60 kg animal andvegetable fats, 770 kg cornsteep liquor, 220 kginorganic compounds (buffer, sources of sulfurand phosphorus), and 100 kg phenylacetic acidas precursors. The amount of product and thedistribution of energy are shown in Table 1.

The energy requirements for the productionof 100 kg of benzylpenicillin sodium are:

Electrical energy: 10.8 GJ (mainly for stirring)Steam: 4 t (sterilization, sealing)Fermentation air: 50 000 m3 (at STP)Cooling water: 900 m3

Waste Materials. The accumulation of sub-stantial amounts of fermentation waste materi-als, such as the fungal mycelium and the culture

Table 1. Balance of energy and materials

Mass, Mass Energykg frac- distri-

tion, bution a,% MJ

Raw materials 2350 – –Products

benzylpenicillin sodium 100 4 2453Fungal mycelium 825 35 11 744Remaining substancein culture medium 660 28 8051Carbon dioxide b 765 33 13 176

Total 35 424a as heat of formation or combustion.b Heat of combustion released during transition to carbon dioxide (carried off with the spent air or cooling water).

Table 2. Fermentation residues used as raw material for further fermentation

Fermentation residue Condition Raw material for subsequent fermentationReferencePenicillium (from benzylpenicillin) moist mycelium oxytetracycline [200]

moist mycelium chlortetracycline [201]moist mycelium streptomycin, vitamin B12, [202]

or riboflavinmycelium hydrolyzate phenoxymethylpenicillin

Penicillium or other mold mycelia mycelium hydrolyzate nutrient medium (e.g., for [203]from fermentation Lactobacillus bifidus)

Penicillium (from benzylpenicillin) moist mycelium calcium gluconate [204]Streptomyces (from tetracyclines) moist mycelium tetracyclines, etc. [205]Penicillium, Aspergillus, Rhizopus, moist mycelium nisin, in connection with lactic [206]

or yeast acid fermentation (silage)Penicillium, Aspergillus, Actinomyces, Rhizopus,yeasts, and activated sludge (from water treatmentplants)

moist or dry mycelium moenomycin (flavophospholipol) [207]

42 Antibiotics

solution freed of antibiotic, is a real problem.After the removal of organic solutions, e.g., bydistillation (stripping), the spent medium mustbe fed into a biological water treatment plant.Seepage is no longer allowed.

The fungal mycelium can be processed or dis-posed of in several ways.

1) It can be fed to animals, directly or after dry-ing. The proceeds cover only a part of thecosts, especially if the mycelium has beendried. Also, the presence of antibiotics in thefilter cake must be avoided, and filtering aids,e.g., kieselguhr or activated charcoal, maynot be used.

2) It can be incinerated after the addition of liq-uid fuel; this is a clean but very expensiveprocedure.

3) It can be disposed of by dumping and hu-mus production along with sludge deposit-ing. This alternative often must be consid-ered, although it entails high transportationcosts.

4) It can be recycled. The mycelium can beused, directly or after intermediate process-ing, as a raw material for further fermen-tation. Mycelium is usually used as a nu-trient for another microorganism. This ap-proach is economically attractive (see Table2 [200 – 207]).

Control of the Fermentation Process. Atregular intervals of several hours, samples ofthe culture solution are withdrawn through thesample port for analysis. Important data are ob-tained by means of chemical, physical, and bio-logical tests. The values are plotted and curvesthat present a good picture of the fermentationprocess are obtained.

The most important analyses are:

1) Determination of the amount of antibiotic.(Biological assay is described on 7.)

2) Determination of the weight of the myceliumas an indication of the growth of the microor-ganism. After inoculation of the fermenterand an initial slow phase, rapid multiplicationoccurs. This slows down later and finally al-most comes to a standstill. The point in timeat which the antibiotic production decreasesand falls short of economic viability can beempirically determined. At about this point

the fermentation is interrupted and the cul-ture harvested.

3) Microscopic control of the growth of the mi-croorganism.

4) Sterility tests, i.e., tests for the absence offoreign microorganisms.

5) Measurement and correction of the pH of theculture.

6) Determination of sugar. Figures 3 A and 3 Bgraphically show two possibilities. Figure 3A shows the sugar consumption. The amountof sugar consumed, plotted against time, isthe difference between the total amount ofsugar added and the analytically determinedsugar concentration at that particular time.Figure 3 B shows the sugar content of the nu-trient solution. Here, the shape of the curveis a measure of the sugar consumption (pro-vided no more sugar is added).

7) Determination of nitrogen in the myceliumand in the culture solution, possibly com-bined with the addition of a nitrogen-con-taining nutrient solution.

6. Isolation and Purification ofAntibiotics; Quality Specifications

6.1. Isolation

When fermentation is completed, i.e., when asufficiently high amount of antibiotic has ac-cumulated in the culture solution, the antibioticmust be separated from the spent medium. Thecontents of the fermenter are transferred to a har-vesting tank so that the fermenter can be turnedimmediately to the production of the next batch.The aeration is stopped, the solution cooled ifnecessary, and recovery of the product is begunas soon as possible. If permitted, a disinfectant,e.g., formaldehyde, is added or heat is applied toprevent further proliferation of the microorgan-ism.

The recovery of the antibiotic can be carriedout in several different ways, depending not onlyon its properties, but also on its subsequent pro-cessing.

Drying Process. Technically speaking, theeasiest and cheapest process is to dry the en-tire culture, the culture filtrate, or the filter cake.Drying is employed on a large scale only in the

Antibiotics 43

Figure 3. A. Penicillin formation with continuous glucose addition after development of the mycelium [208]B. Streptomycin formation by Streptomyces griseus [209]

manufacture of antibiotics used to supplementanimal feed, e.g., tetracyclines and moenomycin(flavophospholipol, flavomycin) or salinomycin.Spray drying is the method most often used.Other methods, such as roller drying (possiblyunder vacuum) are also used. It is advisable toconcentrate the solution, e.g., using a downdraftevaporator, before it is actually dried. In anycase, the antibiotic must be resistant to highertemperatures. Because of its very short heatingtime, the spray-drying method is one of the mostgentle procedures.

Filtration Followed by Extraction and Pre-cipitation. The mycelium is separated from theliquid medium by passing the entire culture so-lution through a filter press, using filtering aids ifnecessary. A rotating filter can also be used e.g.,an Oliver filter, which has three zones, intendedfor suction, washing, and peeling. If the culture

solutions contain small amounts of mycelium,separation can also be carried out in a centrifuge.

Extraction is the method used to separatemost antibiotics contained in the filtrate. A clas-sic example is the extraction of benzylpenicillin(and phenoxymethylpenicillin) with butyl ac-etate (Fig. 4). It leads to a 120- to 150-fold en-richment. The penicillins are then precipitatedfrom the extract as salts. Only those organicbases that preferentially form sparingly solublesalts with penicillin G or V but highly solublesalts with other penicillins can be used for pre-cipitation from either water or organic solvents.

Tertiary morpholines, N-ethylhexahydro-picoline and N-ethylpyrrolidine, besides N-ethylpiperidine, can be used for precipitationfrom butyl acetate, amyl acetate, and similaresters. N-Ethylhexamethyleneimine is used forprecipitation from chloroform.

The salts thus obtained generally are easilycrystallized easily and are quite stable in the dry

44 Antibiotics

Figure 4. Penicillin extraction and the sterile final stagea) Rotameter; b) Mixer

state. They can be stored until further productionsteps are carried out to give the product that isused in clinical practice.

Filtration and Direct Precipitation. Afterfiltration the aqueous culture filtrate can be sub-jected to direct precipitation. This method wasonce important in the isolation of streptomycinas a highly insoluble, colored salt, but this usehas long been abandoned. Direct precipitationfrom the culture filtrate is of interest now be-cause the amount of antibiotic produced by to-day’s highly developed, efficient strains is solarge that the traces of antibiotic remaining in theaqueous solution after precipitation are negligi-ble. This method has acquired importance in theisolation of, e.g., tetracycline, which can be pre-cipitated at its isoelectric point (pH 4.8) [210].Another example is the direct precipitation of5-hydroxytetracycline using a long-chain qua-ternary ammonium salt [211].

Filtration Followed by Extraction from theFilter Cake. If the antibiotic is present entirelyor almost entirely in the mycelium, its isolationis greatly facilitated by filtration, which causes

a considerable decrease in the volume of ma-terial. The moist or dry filter cake bearing theproduct is extracted with a solvent. The resultingsolution is filtered again and processed further[212]. Examples are griseofulvin and moeno-mycin (flavophospholipol, flavomycin).

Adsorption Methods. Adsorption on acti-vated charcoal following filtration is no longerused industrially. However, this method is usedwith some success in the developmental stagesof new antibiotics.

Direct adsorption, e.g., on resins, withoutprior filtration, is still industrially importantfor the separation of such basic antibioticsas streptomycin, kanamycin, neomycin, andparomomycin. Filtration of the culture solution,especially because of the slimy substances pro-duced by actinomycetes, is laborious and re-quireds large amounts of filtering aids. The realbreakthrough came when the adsorption mate-rial (cation exchanger) was brought into contactwith an ascending stream of the culture solution,without prior filtration. In this case the antibioticmolecules leave the solution and attach them-selves to the surface of the adsorption material.

Antibiotics 45

6.2. Purification Techniques, Sterile EndProducts, Official Regulations

Antibiotics are fermentation products and areisolated either as unfinished products or as inter-mediates, generally solid substances of limitedstability. They are purified by methods normallyemployed in organic chemistry, which includechromatography, crystallization, and precipita-tion.

A major requirement is that the antibioticsintended for parenteral administration be freeof pyrogens (fever-producing substances) andhistamine-like compounds. These unwantedsubstances can be carried over from the fermen-tation, but such impurities must not appear inthe final product. Special purification steps, e.g.,treatment with elemental chlorine (destructionof pyrogens associated with streptomycin), fil-tration through activated charcoal, or deep-bedadsorption filtration, must be carried out. Precisetests must confirm the absence of all unwantedsubstances.

In the manufacture, purification, and prepara-tion of antibiotics, special measures also must betaken to prevent penicillin contamination. In theprocessing of active substances to give pharma-ceuticals, a strict spatial separation is necessaryto avoid mutual contamination through the air.

Even minute amounts of antibiotics, espe-cially penicillin, can lead to sensitization in hu-mans and to the formation of resistant microor-ganisms. These antibiotics are then ineffectivein the treatment of diseases because either thepatient is allergic or the pathogens have becomeresistant. Therefore, during the processing andfiltering operations the air supply must be mon-itored very carefully to insure the protection ofthe operating personnel and to guarantee that nopatient unintentionally receives even traces ofpenicillin with another drug.

Production of Sterile Bulk Drug Sub-stances. If sterile bulk drug substances are re-quired, then aseptic conditions must be main-tained from the start of the process. Many anti-biotics are chemically unstable and cannot tol-erate sterilization by heat or other agents. Gen-erally, the solution is sterilized before the fi-nal crystallization, precipitation, spray drying,or freeze drying. This is done by filtering through

porcelain filters, sintered metal filters, layers offilter paper, or graded-porosity films.

Work is carried out in specially equippedsterile rooms, which are fully air conditionedwith practically germ-free air. Air-filtering de-vices similar to those used for the production offermentation air are used. The rooms are disin-fected using, e.g., gaseous formaldehyde, beforework is commenced. The floor is kept damp witha solution of a disinfectant (phenol, quaternaryammonium bases) mixed with glycerol, to con-trol dust. The air pressure in these sterile rooms ishigher than atmospheric pressure. This preventsthe entrance of unclean air. One enters the sterilerooms only after carefully washing and donningsterile clothes and through an airlock equippedwith UV lamps and foot mats soaked in disin-fectant. Small objects (tools, etc.) are broughtinto the sterile room through smaller air locks,in which they are disinfected using intense UVradiation. Containers, e.g., stainless steel cans,are first washed thoroughly and then sterilizedin an autoclave. The autoclave is provided withone door that leads to the unsterile washing roomand a second door leading to the sterile room.

The different steps in the course of furtherprocessing the antibiotics, such as centrifuga-tion, drying, pulverization, sieving, and packag-ing, are performed in sterile glove boxes, whichare provided with sterile air at a slightly elevatedpressure and are equipped with UV irradiators.

Laminar-Flow Technique. In this tech-nique, only one part of a clean or sterile roomis maintained as a clean bench. This area issurrounded by a sterile box provided with aworking access. To avoid the penetration ofunclean air into this confined clean space, a dis-placing, turbulence-free air stream is created inthe box. A continuous stream of sterile, filteredair enters the box from the top or from one sideat a fixed speed. This air is distributed uniformlyand is then sucked out the opposite side at thesame speed. Special devices insure that there isminimum turbulence in the area of contact bet-ween the flowing, sterile air and the stationary,unclean air. Laminar-flow (LF) units can also beused for work with substances that should notescape to the outside world. In this case the airis recirculated through a filter.

46 Antibiotics

Clean Packing. Antibiotics packed in bulk,in large containers, and intended for therapeu-tic use, usually are present in a sterile, highlypure state. Products such as tetracycline hydro-chloride, intended for oral administration, mustbe very pure but not necessarily sterile.

Official Quality Requirements, Pharma-copoeias. The production process and the qual-ity of the end product are subjected to rig-orous official controls. The requirements havelong been stipulated in the pharmacopoeia ofeach country and generally possess legal au-thority. The pharmacopoeias European Phar-macopoeia, International Pharmacopoeia, andCompendium Medicamentorum (standard phar-macopoeia for all Comecon states) are each validin several countries.

In the United States, the influence exerted bythe federal government goes beyond the determi-nation of minimum quality standards for drugs.The Food and Drug Administration (FDA), at-tached to the U.S. Department of Health and Hu-man Services, has published a Code of FederalRegulations (CFR) that is continuously supple-mented with new regulations and reissued annu-ally. The demands on the quality of a drug aresubstantially stricter and more comprehensivethan those stipulated in the U.S. Pharmacopeia.In addition, detailed requirements have been es-tablished for the production and encapsulationtechniques and rooms, the documentation, andthe storage of raw materials, additives, interme-diates, and end products. A detailed analysis ofthe starting materials, process controls, and testsof the end products and preparations must alsobe carried out. When a product not yet approvedby the FDA is to be registered, preliminary testsmust be conducted as well.

Food and Drug Administration officials havethe right to inspect production plants regularly.Complaints can lead to the temporary closingof the plant and to the recall of certain prepa-rations or particular batches of a preparation.In order to achieve a uniform standard in theproduction of drugs, the FDA [213] and theWHO [214 – 217] have elaborated and pub-lished fundamental rules that are now interna-tionally called Regulations for Good Manufac-turing Practices (GMPs). Drugs manufactured incountries outside the United States but importedinto the United States are also subject to FDA

regulations, including the GMPs, and FDA in-spections. The FDA has published detailed rulesin the Federal Regulations [218], especially forthe registration of imported products.

Officials in other countries also demand adetailed description of the manufacture, qual-ity, and safety of any drug they import. In manycases, sales depend on a prerequisite inspectionof the factory, similar to the one conducted bythe FDA. Canadian officials, American militaryforces purchasers, and the British Department ofHealth and Social Security all have this require-ment.

7. Analytical Measurements andQuality Control

The analyses of antibiotics can be divided intotwo basic groups:

1) Tests during production, usually processsurveillance and control.

2) Quality control, practiced as required by theWHO and the FDA. These tests have beenroutinely conducted by independent labora-tories for a long time.

The end product, raw materials, and inter-mediates all are tested. The analytical measure-ments can be divided into chemical and physi-cal tests, on the one hand, and biological tests,on the other. For the former, general methodsused in synthetic organic chemistry are applied,along with some special methods that have beenworked out for antibiotics. Fully or partially au-tomatic techniques have been introduced to han-dle the large number of samples.

7.1. Microbiological Analysis

Biological Assay. Numerous microbiologi-cal methods are available to determine theamount of antibiotic present in a sample.

Agar Diffusion Test (Cylinder-PlateMethod). See [219] for the original method; im-provements are described in [220, 221].

A standard method has been published bythe FDA [222]. For supplements, see [223] and[224].

Antibiotics 47

Twenty milliliters of nutrient agar is placedin a flat-bottomed petri dish. After this solidi-fies, four milliliters of a second nutrient solution,seeded with the test bacteria, is poured evenlyonto the first layer (at 48 ◦C). As soon as thesecond layer has solidified, six sterile stainlesssteel cylinders are placed on the agar, preferablyusing a cylinder-placing machine. To the opencylinders are added equal amounts of a standardpenicillin solution containing 2.0, 1.5, 1.0, 0.5,and 0.25 U/mL. Samples of the test antibiotic so-lution are deposited analogously on other petridishes.

The dishes are incubated at 37 ◦C for 16 –18 h. During this time the penicillin diffuses outof the cylinders into the surrounding agar andsuppresses the growth of the test organism. Thus,the cylinders are surrounded by clear zones, freeof bacteria. The diameter of each zone providesan index of the activity of the penicillin prepa-ration. The mean values obtained from 10 – 20standard plates are used to draw a calibrationcurve, and the biological activity of the test so-lution in international units is determined usingconversion tables.

Antibiotic Disk Method. This is a modifica-tion of the diffusion test. The method is widelyused to determine whether a definite strain or amixture of different microorganisms is sensitiveor resistant to a given antibiotic [225 – 230].

The pathogen is freshly isolated from patientsand used to inoculate a suitable nutrient agarplate. Filter paper disks 6 or 9 mm in diameterare placed on the petri dish before incubation.These disks are impregnated with a solution ofthe antibiotic. The amount is chosen so that theconcentration of active substance present afterdiffusion into the agar medium corresponds tothe level attainable in the patient (blood or tis-sue level). Test doses of 0.5 to 20 U are normalfor a disk test of penicillins G or V.

In order to maintain a certain uniformityin the production of the nutrient medium forthe disk method, the following directions forthe preparation of peptone – casein agar haveproved useful:

Peptone 6.0 gPancreatic – digested casein 4.0 gYeast extract 3.0 gMeat extract 1.5 gDextrose 1.0 gAgar 15.0 g

The components are dissolved in 1000 mLof distilled water and the pH of the liquid agaris adjusted to 6.55 after sterilization. If an agarplate thus prepared is incubated, the growth ofthe microorganisms seeded on the plate can beobserved from the turbidity of the agar surface.If the antibiotic on the disk is effective, a clearzone of inhibition forms surrounding the disk(Fig. 5). Table 3 shows the experimental valuesfor the diameters of the zones of inhibition forStaphylococcus aureus ATTC 6538P, using thepH 6.55 nutrient medium described above.

Figure 5. Antibiotic disk testThe diameter of the clear zone depends on the test dose ofantibiotic: disk P (left) contained 0.5 U, disk *P (right) 20U benzylpenicillin sodium.

Table 3. Diameter of zones of inhibition for Staphylococcus aureusATTC 6538 P

Antibiotic Concentration Inhibitionper disk zone

Ampicillin 5 µg 26Chloramphenicol 10 µg 20Lincomycin 2 µg 19Methicillin 10 µg 26Novobiocin 10 µg 26Oxacillin 10 µg 30Penicillin (P) 0.5 I.U. 26Penicillin (* P) a 20 I.U. >40Streptomycin b 10 µg 16Tetracycline 10 µg 27a Massive dose of penicillin (see Fig. 5);b A pH of 8.0 is required for the optimal evaluation of thesubstance. Under these conditions the diameter of this zone is also26 mm.

48 Antibiotics

Tube Dilution Method. Three milliliters ofa nutrient solution is put into each of a row oftubes. Three milliliters of a penicillin solutionwith a dilution of 1 : 100 is pipetted into the firsttube. After thorough mixing, three millimeters isremoved and added to the next tube. After mix-ing, three millimeters is removed and added tothe third tube, and so on. The tubes contain suc-cessively lower concentrations of the drug. If theinitial penicillin solution had a 1 : 100 dilution,the first tube now contains a 1 : 200 dilution, thesecond a 1 : 400, and so on.

Each tube is inoculated with one drop of aday-old staphylococcus culture (test bacteria).After incubation for one day at 37 ◦C, the endpoint is determined, i.e., the lowest concentra-tion of penicillin that prevents the developmentof turbidity. If tube 3, for example, is clear buttube 4 is turbid, the end point is calculated bymultiplying the starting dilution of 1 : 100 by 23.Thus the bacteriostatic units are obtained, here800 Bact. U. These can be converted into in-ternational units with the help of the penicillinsensitivity factor. For instance, if this factor is0.04, then 25 Bact. U = 1 I. U. and the solutioncontains 800/25 = 32 I. U. [231 – 234].

Slight turbidity also can be caused by proteinprecipitation resulting from a change in pH. Forthis reason, it is advisable to add a pH indica-tor to the nutrient broth. Changes in the pH arethen clearly visible. Phenol red and bromothy-mol blue generally are used. A critical assess-ment of the test is given in [223].

The tube dilution method is also used to de-termine the sensitivity of freshly isolated singlestrains or mixtures of pathogens to antibiotics. Acomparison of the lowest inhibitory concentra-tion of an antibiotic with the serum levels attain-able in vivo indicates which antibiotic is mostsuitable for clinical administration.

Automatic Analyses. Automatic sample re-moval and preparation have speeded up anal-yses. However, in cases that involve specialpreparatory steps, such as dissolution, filtration,or extraction, full automation is not yet possible.Many references discussing automatic biotest-ing, are available [223, 224, 235 – 240].

The measurement of the clear zones in theagar diffusion test, which formerly was con-ducted visually, can now be performed objec-tively and automatically using commercially

available instruments (scanning analysis sys-tems). By connecting a laboratory calculator(e.g., HP 85) to such an instrument, the amountof antibiotic in a test solution can be calculatedusing reference standards and printed out di-rectly.

7.2. Isotopically Labeled Antibiotics

Antibiotics containing a radioisotope at a defi-nite position in the molecule are very importantfor scientific studies. Labeled substances can beused to trace:

1) Accumulation in specific tissues, e.g., tu-mors, for diagnostic purposes.

2) Metabolism of an antibiotic, i.e., tracing themetabolites and cleavage products in animaland human organs or excretions.

3) Determining the location and partial degra-dation of the antibiotic during further fer-mentation, processing, and purification steps.

Isotopically labeled antibiotics can be manu-factured in two ways.

Fermentation Production. If appropriateisotopically labeled compounds are added to theculture solution during fermentation, a corre-sponding amount of labeled antibiotic is pro-duced and can be isolated with the unlabeledantibiotic. This technique also is suitable for fol-lowing labeled precursors, nutrients, salts, etc.,during the fermentation procedure. In this way,insights into the mechanism of formation ofthe antibiotic in a microorganism or the mech-anism of formation in the presence of an iso-lated enzyme system can be obtained. Ben-zylpenicillin, phenoxymethylpenicillin, and 6-aminopenicillanic acid have been labeled with14C and 35S in this way [210, vol. 4, pp. 266,296], [241]. Streptomycin has been labeled with14C and 3H [210, vol. 4, p. 349].

Labeling. Acylation of 6-aminopenicillanicacid with a 14C-labeled acid yields a product la-beled in the side chain [242]. A subsequent iso-topic exchange within the antibiotic molecule isalso possible. However, only the easily removed1H atoms can be replaced by 2H or 3H.

Antibiotics 49

8. Economic Aspects

Antibiotics find widespread use in human andveterinary medicine. As yet, agricultural usageis low and generally confined to Asia wherethey are used for antifungal treatment of riceplants, etc. Over 30 kt/a of antibiotics was pro-duced worldwide in 1984. There are six maincategories of antibiotics:

1) β-Lactam2) Tetracycline3) Macrolide4) Peptide and glycopeptide5) Aminoglycoside6) Polyether

At least twenty other commercial antibioticsare not included within these six categories.They belong to a variety of chemical types, i.e.,polyene, ansamycin, anthracycline, nucleoside,etc.

Over the past ten years, output has grownby approximately 4 % per year, with the mostrapid growth in the β-lactams, macrolides, andpolyethers. On the other hand, tetracyclines havepresented a static or declining market, partic-ularly for human therapy. Dollar volume saleshave grown correspondingly, with a successfulnew human antibiotic product being defined asone commanding minimal sales of $ 100 000000 worldwide. β-Lactam sales account for atleast half of the total human antibiotic market,which exceeds $ 5 000 000 000.

All the categories except the polyethers finduse in human medicine. The most important vet-erinary antibiotics belong to the tetracycline,macrolide, peptide, and polyether families al-though some β-lactams, aminoglycosides, andother antibiotics also have veterinary markets.

Worldwide, there are over seventy primaryproducers of antibiotics by fermentation. Ifcompanies involved in producing semisyntheticpenicillins and cephalosporins from purchasedparent antibiotics are included, the number iswell over one hundred. Some companies spe-cialize in the production of a single antibi-otic, but more generally a number of differ-ent antibiotics are produced, e.g., benzylpeni-cillin, phenoxymethylpenicillin, cephalosporinC, oxytetracycline, and streptomycin. Largemultinational pharmaceutical companies fre-quently operate a number of separate antibiotic

fermentation plants in one or more countries. Fortechnical reasons, it may not be possible to pro-duce two different antibiotics in the same plant.

United States and European companies areactive in all categories of antibiotics; Japanese,Chinese, and Korean producers have tended tospecialize in the aminoglycosides, macrolides,anticancer drugs, semisynthetic second- andthird-generation β-lactams, and agriculturalantibiotics.

Some old antibiotics, which are no longerprotected by patents, are traded in bulk at theprices quoted in the following paragraphs. Thebulk products are purchased for use in special-ities or conversion into semisynthetic drugs bycompanies that do not have their own fermenta-tion facilities or whose fermentation capacitiesare not adequate to supply their growing needs.Bulk antibiotics are also purchased on tender bygovernment agencies, charities, etc., for use indeveloping countries. There is usually a signifi-cant difference between a bulk price and that ofa finished (branded or generic) speciality.

In the following paragraphs the estimatedworldwide antibiotic output for the year 1985is listed. The antibiotics are grouped into thesix main categories plus “other antibiotics.” Theproduction figures include antibiotics for humanand veterinary applications. The specific com-pounds are arranged alphabetically, not accord-ing to their commercial importance. The bulkprices are quoted only for the antibiotics that aretraded; this price is much lower than the price ofthe finished specialty product.

β-Lactams. Total output is 10 – 20 kt/a.There are over 50 producers. The bulk pricefor benzylpenicillin is 25 – 30 $/kg. The follow-ing compounds are included: ampicillin, amoxy-cillin, carbenicillin, cefaclor, cefamandole, cefa-zolin, cefoperazone, cefotaxime, cefoxitin, cef-tazidime, cefuroxime, cephadroxil, cephalexin,cephalosporin C, cephalothin, cephamycin C,cephradine, clavulanic acid, cloxacillin, di-cloxacillin, flucloxacillin, oxacillin, benzylpeni-cillin, phenoxymethylpenicillin, piperacillin,and ticarcillin.

Tetracyclines. Total output is 5 – 10 kt/a.There are 30 – 40 producers. The bulk pricefor oxytetracycline is 25 – 30 $/kg. The fol-lowing compounds are included: chlortetra-

50 Antibiotics

cycline, democlocycline, doxycycline, metha-cycline, minocycline, oxytetracycline, and tetra-cycline.

Macrolides. Total output is 3 – 5 kt/a. Thereare 20 – 30 producers. The bulk price forerythromycin base is 100 – 120 $/kg. The fol-lowing compounds are included: erythromycin,ivermectin, josamycin, kitasamycin, mideca-mycin, milbemycin, miocamycin, oleando-mycin, spiramycin, and tylosin.

Peptides and Glycopeptides. Total outputis 2 – 3 kt/a. There are 10 – 20 producers. Thebulk price for bacitracin is 15 $/kg. The follow-ing compounds are included: avoparcin, bac-itracin, colistin, enramycin, gramicidin, nisin,polymixin, and thiopeptin.

Aminoglycosides. Total output was 1 – 2kt/a. There are 20 – 30 producers. The bulkprice for streptomycin is 30 $/kg. The follow-ing compounds are included: amikacin, apra-mycin, dibekacin, dihydrostreptomycin, gen-tamicin, hygromycin, kanamycin, lincomycin,neomycin, netilmicin, paromomycin, ribosta-mycin, sagamicin, sisomicin, streptomycin, andtobramycin.

Polyethers. Total output is 3 – 5 kt/a. Thereare 5 – 10 producers. The following compoundsare included: laidlomycin, lasalocid, maduro-mycin, moenomycin, monensin, narasin, andsalinomycin.

Other Antibiotics. Total output was 1 – 2kt/a. There are 30 – 40 producers. The follow-ing compounds are included: amphotericin, an-ticancer (including bleomycin, daunorubicin,doxorubicin, epirubicin, and mitomycin), blas-ticidin, clindamycin, cycloserine, flavomycin,fusidic acid, griseofulvin, novobiocin, nys-tatin, pimaricin, pleuromutilin, pyrrolnitrin,rifampicin, spectinomycin, vancomycin, vio-mycin and virginiamycin.

9. References

General References1. D. Gottlieb, P. D. Shaw (eds.): Antibiotics, vol.

I. Mechanism of Action, Springer-Verlag,Berlin – Heidelberg – New York 1967.

2. D. Gottlieb, P. D. Shaw (eds.): Antibiotics, vol.II, Biosynthesis, Springer-Verlag, Berlin –Heidelberg – New York 1967.

3. J. W. Corcoran, F. E. Hahn (eds.): Antibiotics,vol. III, Mechanism of Action of Antimicrobialand Antitumor Agents, Springer-Verlag, Berlin– Heidelberg – New York 1975.

4. J. W. Corcoran (ed.): Antibiotics, vol. IV,Biosynthesis, Springer-Verlag, Berlin –Heidelberg – New York 1981.

5. F. E. Hahn (ed.): Antibiotics, vol. V, part 1,Mechanisms of Action of Antibiotic Agents,Springer-Verlag, Berlin – Heidelberg – NewYork 1979.

6. F. E. Hahn (ed.): Antibiotics, vol. V, part 2,Mechanisms of Action of Antieukaryotic andAntiviral Compounds, Springer-Verlag, Berlin– Heidelberg – New York, 1979.

7. J. S. Glasby: Encyclopedia of Antibiotics, 2nded., J. Wiley & Sons, Chichester – New York– Brisbane – Toronto 1979.

8. G. Lancini, F. Parenti: Antibiotics. AnIntegrated View, Springer-Verlag, New York –Heidelberg – Berlin 1982.

9. L. P. Garrod, H. P. Lambert, F. O’Grady:Antibiotic and Chemotherapy, ChurchillLivingstone, Edinburgh – London –Melbourne – New York 1981.

10. E. F. Gale, E. Cundliffe, P. E. Reynolds, M. H.Richmond, M. J. Waring: The Molecular Basisof Antibiotic Action, 2nd ed., J. Wiley & Sons,London – New York – Sydney – Toronto 1981.

11. Kirk-Othmer, 3rd ed., vol. 2, 3.12. J. Berdy, A. Aszalos, M. Bostian, K. L.

McNitt, CRC Handbook of AntibioticCompounds, vol. I – X, CRC Press Inc., BocaRaton, Florida, 1980.

13. H. Umezawa (ed.): Index of Antibiotics fromActinomycetes, vol. I, II, Japan ScientificSocieties Press, Tokyo, University Park Press,Baltimore 1978.

14. T. Korzylski, Z. Kowszyk-Gindifer, W.Kurylowicz: Antibiotics Origin, Nature, andProperties, American Society forMicrobiology, Washington D.C., 1978.

15. M. J. Weinstein, G. H. Wagman: “AntibioticsIsolation, Separation and Purification,” J.Chromatogr. Libr. 15 (1978) .

16. R. Reiner: Antibiotics, Georg Thieme Verlag,Stuttgart 1982.

17. W. Kurylowicz (ed.): Antibiotics (a CriticalReview), Polish Medical Publishers, Warsaw1976.

Antibiotics 51

18. P. Sammes (ed.): Topics in AntibioticChemistry, vol. I – VI, Ellis Horwood Ltd,1980.

19. H. P. Kuemmerle (ed.): ClinicalChemotherapy, vol. I, II, III, Thieme-StrattonInc., New York 1984.

Specific References20. S. A. Waksman, Antibiot. Chemother. (Basel,

1954 – 70) 6 (1956) 90.21. S. A. Waksman, The Antibiotic Era, The

Waksman Foundation of Japan Inc., Tokyo1975.

22. A. Fleming, Br. J. Exp. Pathol. 10 (1929) 226.23. G. Domagk, Dtsch. Med. Wochenschr., 61

(1935) 250.24. E. B. Chain et al., Lancet II (1940) 226.25. E. P. Abraham, P. B. Loder, in E. H. Flynn

(ed.): Cephalosporins and Penicillins,Academic Press, New York 1972, p. 1.

26. W. Durckheimer et al., Angew. Chem. Int. Ed.Engl. 24 (1985) 180.

27. R. D. G. Cooper in P. G. Sammes (ed.): Topicsin Antibiotic Chemistry, vol. 3, Ellis HorwoodLimited, Chichester 1980, p. 39.

28. F. A. Jung, W. R. Pilgrim, J. P. Poyser, P. J.Siret in P. G. Sammes (ed.): Topics inAntibiotic Chemistry, vol. 4, Ellis HorwoodLimited, Chichester 1980, p. 11.

29. G. Albers-Schonberg et al. in R. B. Morin, M.Gorman (eds.): Chemistry and Biology ofβ-Lactam Antibiotics, vol. 1, vol. 2, vol. 3,Academic Press, New York 1982.

30. L. P. Garrod, H. P. Lambert, F. O’Grady:Antibiotic and Chemotherapy, 4th ed.,Churchill Livingstone, Edinburgh 1973.

31. S. A. Waksman, Jpn. J. Microbiol. 10 (1966)129.

32. H. Umezawa: Enzyme Inhibitors of MicrobialOrigin, University of Tokyo Press, Tokyo1972.

33. E. P. Abraham in S. Mitsuhashi (ed.):Beta-Lactam Antibiotics, Japan ScientificSocieties Press, Tokyo, and Springer-Verlag,Berlin – Heidelberg – New York 1981, p. 3.

34. D. C. Hodgkin, Advancemt Sci. 6 (1949) 85.35. J. C. Sheehan, K. R. Henery-Logan, J. Am.

Chem. Soc. 79 (1957) 1262;81 (1959) 3089.

36. S. Nakatsuka, H. Tanino, Y. Kishi, J. Am.Chem. Soc. 97 (1975) 5008, 5010.

37. J. E. Baldwin, M. A. Christie, S. B. Haber, L.I. Kruse, J. Am. Chem. Soc. 98 (1976) 3045.

38. R. B. Woodward et al., J. Am. Chem. Soc. 88(1966) 852.

39. E. O. Stapley, J. Birnbaum in M. Salton, G. D.Shockman (eds): β-Lactam Antibiotics,Academic Press, New York 1981, p. 327.

40. S. Karady et al., Tetrahedron Lett. 1976, 2401.41. S. Karady et al., J. Am. Chem. Soc. 94 (1972)

1410.42. L. M. Weinstock et al. Tetrahedron Lett. 1975,

3979.43. M. Shinozaki, N. Ishida, K. Iino, T. Hiraoka, J.

Chem. Soc. Chem. Commun. 1978, 517.44. Y. Hamashima, H. Matsumura, S. Matsuura,

W. Nagara, M. Narisada, T. Yoshida in G. I.Gregory (ed.): Recent Advances in theChemistry of β-Lactam Antibiotics, SpecialPubl. no. 38, The Royal Society of Chemistry,London 1981, p. 57.

45. W. Nagata in H. Nozaki (ed.): Current Trendsin Organic Synthesis, Pergamon Press, Oxford1983, p. 83.

46. K. Shimizu, Japanese J. Antibiot. 41 (1988)1809.

47. A. G. Brown et al., J. Antibiot. 29 (1976) 668.48. N. Aswapokee, H. C. Neu, J. Antibiot. 31

(1978) 1328.49. P. Raillard, C. Feiner, V. Ott, G. Treadway, Y.

Wang, Current Therapeutic Res.- Clinical andExperimental 55 (1994) 601.

50. F. Mooosdeen, J. D. Williams, S. Yamabe,Antimicrob. Agents Chemother. 32 (1988) 925.

51. M. Ishiguro, T. Nishihara, R. Tanaka,Yakugaku Zasshi 121 (2001) 915.

52. A. G. Brown et al., in J. Elks (ed.): RecentAdvances in the Chemistry of β-LactamAntibiotics, Special Publ. no. 28, TheChemical Society, London 1977, p. 295.

53. J. S. Kahan et al., Intersci. Conf. Antimicrob.Agents Chemother., 16th, Chicago, Pap. no.227 (1976).

54. M. Barza, Ann. Intern. Med. 103 (1985) 552.55. M. Fukasawa, Y. Sumita, E. Tada, T. Okuda,

Chemoterapy 40 (1992) 74.56. C. R. Catchpole, R. Wise, D. Thomber, J. M.

Andrews, Antimicrob. Agents Chemother. 36(1992) 1928.

57. Y. Utsui et al., Chemotherapy 39 (1991) 83.58. H. Aoki et al., J. Antibiot. 29 (1976) 492.59. A. Imada, K. Kitano, K. Kintaka, M. Muroi,

M. Asai, Nature (London) 289 (1981) 590.60. R. B. Sykes et al. Nature (London) 291 (1981)

489.61. C. M. Cimarusti et al., J. Org. Chem. 47

(1982) 179.62. A. Imada, M. Kondo, K. Okonogi, Antimicrob.

Agents Chemother. 27 (1985) 821.63. B. M. Duggar, Ann. N.Y. Acad. Sci. 51 (1948)

177.

52 Antibiotics

64. F. Arcamone, A. D. Di Marco, M. Gaetani, T.Scotti, G. Microbiol. 9 (1961) 83.

65. F. Arcamone in P. G. Sammes (ed.): Topics inAntibiotic Chemistry, vol. 2, Ellis HorwoodLtd., Chichester 1978, p. 99.

66. S. Neidle in P. G. Sammes (ed.): Topics inAntibiotic Chemistry, vol. 2, Ellis HorwoodLtd., Chichester 1978, p. 240.

67. S. A. Waksman: Streptomycin, The Williamsand Wilkins Co., Baltimore 1949;Neomycin, Rutgers Univ. Press, NewBrunswick, New Jersey, 1953.

68. L. Weinstein, N. J. Ehrenkranz in: H. Welch, F.Marti-Ibanez (eds.): Streptomycin and DHS,Antibiotic Monographs, no. 10, MedicalEncyclopedia, New York 1958.

69. H. Schmidt et al., Pharmazie 23 (1968) 161.70. J. S. Brimacombe, Angew. Chem. 83 (1971)

261.71. H. Grisebach, R. Schmid, Angew. Chem. 84

(1972) 192.72. H. Umezawa et al., J. Antibiot. 24 (1971) 485.73. J. Davies, D. I. Smith in M. Starr (ed.): Annual

Review of Microbiology, vol. 32, AnnualReviews Inc., California 1978, p. 469.

74. H. Umezawa in R. S. Tipson, D. Horton (eds.):Advances in Carbohydrate Chemistry andBiochemistry, vol. 30, Academic Press, NewYork 1974, p. 183.

75. H. Umezawa, in S. Mitsuhashi (ed.): DrugAction and Drug Resistance in Bacteria, vol.2, University of Tokyo Press, Tokyo 1975, p.211.

76. H. Umezawa, S. Kondo in H. Umezawa, I. R.Hooper (eds.): Handbook of ExperimentalPharmacology, vol. 62, Springer Verlag,Heidelberg 1982.

77. R. U. Lemieux, M. L. Wolfrom in W. W.Pigman, M. L. Wolfrom (eds.): Advances inCarbohydrate Chemistry, vol. 3, AcademicPress, New York 1948, p. 337.

78. S. Neidle, D. Rogers, M. B. Hursthouse,Tetrahedron Lett. 1968, 4725.

79. S. Umezawa et al., J. Antibiot. 27 (1974) 997.80. A. Bloch in E. J. Ariens (ed.): Drug Design,

vol. 4, Academic Press, New York 1973, p.286.

81. M. Ohno in J. M. Cassady, J. D. Douros (eds.):Anticancer Agents Based on Natural ProductModels, Academic Press, New York 1980, p.73.

82. A. Bloch in M. Grayson (ed.): Antibiotics,Chemo-therapeutics, and Antibacterial Agentsfor Disease Control, J. Wiley & Sons, NewYork 1982.

83. K. Mizuno, M. Tsujino, M. Takada, M.Hayashi, K. Atsumi, K. Asano, T. Matsuda, J.Antibiot. 27 (1974) 775.

84. T. Tsuruoka et al., Meiji Seika Kenkyu Nempo9 (1967) 17.

85. P. W. K. Woo, H. W. Dion, et al., J. Heterocycl.Chem. 11 (1974) no. 4, 641 – 3.

86. H. Nakamura, G. Koyama, Y. Iitaka, M. Ohno,N. Yagisawa, S. Kondo, K. Maeda, H.Umezawa, J. Am. Chem. Soc. 96 (1974) 4327.

87. K. Isono, K. Asahi, S. Suzuki, J. Am. Chem.Soc. 91 (1969) 7490.

88. K. Isono, S. Suzuki, Heterocycles 13 (1979)333.

89. M. Uramoto, M. Matsuoka, J. G. Liehr, J. A.McCloskey, K. Isono, Agric. Biol. Chem. 45(1981) 1901.

90. M. Hori, E. Ito, T. Takita, G. Koyama, T.Takeuchi, H. Umezawa, J. Antibiot. Ser. A 17(1964) 96.

91. S. Aizawa, T. Hidaka, N. Otake, H. Yonehara,K. Isono, N. Igarashi, S. Suzuki, Agric. Biol.Chem. 29 (1965) 375.

92. G. Koyama, H. Umezawa, J. Antibiot. Ser. A.18 (1965) 175.

93. H. Nishimura, M. Mayama, Y. Komatsu, H.Kato, N. Shimaoka, Y. Tanaka, J. Antibiot. Ser.A 17 (1964) 148.

94. T. Kusaka, H. Yamamoto, M. Shibata, M.Muroi, T. Kishi, K. Mizuno, J. Antibiot. 21(1968) 255.

95. S. Yaginuma, N. Muto, M. Tsujino, Y. Sudate,M. Hayashi, M. Otani, J. Antibiot. 34 (1981)359.

96. M. Arita, K. Adachi, Y. Ito, H. Sawai, M.Ohno, J. Am. Chem. Soc. 105 (1983) 4049.

97. S. Takeuchi et al. J. Antibiot. Ser. A 11 (1958)1.

98. S. Masamune, G. S. Bates, J. W. Corcoran,Angew. Chem. Int. Ed. Engl. 16 (1977) 585.

99. S. Masamune, J. W. Ellingboe, W. Choy, J.Am. Chem. Soc. 104 (1982) 5526.

100. M. N. Donin et al., Antibiot. Annu. 1953 –1954, 179.

101. C. Djerassi, J. A. Zderic, J. Am. Chem. Soc. 78(1956) 2907.

102. T. Hata et al., J. Antibiot. Ser. A 6 (1953) 87.103. S. Pinnert-Sindico et al., Antibiot. Annu. 2

(1954 – 55) 274.104. R. B. Morin, M. Gorman, P. L. Hamill, P. V.

Demarco, Tetrahedron Lett. 1970, 4737.105. S. Bhuwapathanapun, P. Gray, J. Antibiot. 30

(1977) 673.

Antibiotics 53

106. S. Omura, H. Matsubara, A. Nakagawa, A.Furusaki, T. Matsumoto, J. Antibiot. 33 (1980)915.

107. F. J. Antosz in M. Grayson (ed.): Antibiotics,Chemotherapeutics and Antibacterial Agentsfor Disease Control, J. Wiley & Sons, NewYork 1982, p. 76.

108. K. L. Rinehart, Jr. Acc. Chem. Res. 5 (1972)57.

109. P. Sensi, Res. Prog. Org. Biol. Med. Chem. 1(1964) 337.

110. M. T. Timbal, Antibiot. Annu. 1959 – 1960,271.

111. N. Bergamini, G. Fowst, Arzneim. Forsch. 15(1965) 951.

112. S. Riva, L. G. Silvestri, Annu. Rev. Microbiol.26 (1972) 199.

113. D. Perlman in M. Grayson (ed.): Antibiotics,Chemotherapeutics, and Antibacterial Agentsfor Disease Control, J. Wiley & Sons, NewYork 1982, p. 210.

114. H. Umezawa, K. Maeda, T. Takeuchi, Y.Okami, J. Antibiot. Ser. A. 19 (1966) 200.

115. T. Takita, Y. Muraoka, T. Nakatani, A. Fujii, Y.Umezawa, H. Naganawa, H. Umezawa, J.Antibiot. 31 (1978) 801.

116. T. Takita et al., Tetrahedron Lett. 1982, no. 23,521, and references cited therein.

117. H. Umezawa in J. M. Cassady, J. D. Douros(ed.): Anticancer Agents Based on NaturalProduct Models, Academic Press, New York1980, p. 147.

118. G. F. Gauze, M. G. Brazhnikova, Lancet 247(1944) 715.

119. C. Ressler, D. V. Kashelikar, J. Am. Chem.Soc. 88 (1966) 2025.

120. G. M. Schull, J. L. Sardinas, Antibiot.Chemother. 5 (1955) 398.

121. A. E. Oxford, H. Raistrick, P. Simonart,Biochem. J. 33 (1939) 240.

122. J. F. Grove, J. MacMillan, T. P. C. Mulholl, M.A. T. Rogers, J. Chem. Soc. 1952, 3977.

123. J. Ehrlich, Q. R. Bartz, R. M. Smith, D. A.Joslyn, P. R. Burkholder, Science (WashingtonD.C.) 106 (1947) 417.

124. G. Keiser, Dtsch. Med. Wochenschr. 96 (1971)1544.

125. K. Shirahata, N. Hirayama, J. Am. Chem. Soc.105 (1983) 7199.

126. T. Hata, Y. Sano, R. Sugawara, A. Matsume,K. Kanamori, T. Shima, T. Hoshi, J. Antibiot.Ser. A 9 (1956) 141.

127. S. Wakaki, H. Marumo, K. Tomioka, G.Shimizu, E. Kato, H. Kamada, S. Kudo, Y.Fujimoto, Antibiot. Chemother. (WashingtonD.C.) 8 (1958) 228.

128. D. Hendlin et al., Science (Washington D.C.)166 (1969) 122.

129. W. O. Godtfredsen, S. Jahnsen, H. Lorck, K.Roholt, L. Tybring, Nature (London) 193(1962) 987.

130. B. Guignard, J. M. Entenza, P. Moreillon,Curr. Opinion Pharmacol. 5 (2005) 479.

131. K. C. Nicolaou, W.-M. Dai, Angew. Chem. Int.Ed. Engl. 30 (1990) 1387.

132. N. Ishida, K. Miyazaki, K. Kumagai, M.Rikimaru, J. Antibiot. 18 (1965) 68.

133. K. Edo et al., Tetrahedron Lett. 26 (1985) 331.134. M. D. Lee et al., J. Am. Chem. Soc. 109 (1987)

3464.135. M. D. Lee et al., J. Am. Chem. Soc. 109 (1987)

3466.136. J. Golik et al., J. Am. Chem. Soc. 109 (1987)

3461.137. J. Golik et al., J. Am. Chem. Soc. 109 (1987)

3462.138. M. Konishi et al., J. Antibiot. 42 (1989) 1449.139. A. G. Meyer, Tetrahedron Lett. 28 (1987)

4493.140. V. H. J. Vander Velden et al., Blood 97 (2001)

3197.141. L. M. Hinmar, P. R. Hamann, R. Wallace, A. T.

Menendez, F. E. Durr, J. Upeslacis, CancerRes. 53 (1993) 3336.

142. H. Maeda, K. Edo, N. Ishida (eds.):Neocarzinostatin. The Past, Presend, andFuture of an Anticancer Drug,Springer-Verlag, Heidelberg 1997.

143. J. H. Boothe et al., J. Am. Chem. Soc. 75(1953) 4621.L. H. Conover et al., J. Am. Chem. Soc. 75(1953) 4622.

144. J. R. D. McCormick et al., J. Am. Chem. Soc.79 (1957) 4561.

145. A. C. Finlay et al., Science (Washington, D.C.)111 (1950) 85.P. P. Regna, I. A. Solomons, Ann. N.Y. Acad.Sci. 53 (1950) 221.

146. C. R. Stephens et al., J. Am. Chem. Soc. 80(1958) 5324.J. R. D. McCormick et al., J. Am. Chem. Soc.82 (1960) 3381.

147. G. S. Redin, Antimicrob. Agents Chemother.(1961 – 70) 1967, 371.

148. A. D. Di Marco et al., Nature (London) 201(1964) 706.F. Arcamone et al., J. Am. Chem. Soc. 86(1964) 5334.

149. F. Arcamone et al., Tetrahedron Lett. 1969,1007.

54 Antibiotics

150. T. Oki et al., J. Antibiot. 28 (1975) 830.151. H. Umezawa et al., J. Antibiot. Ser. A 10

(1957) 181.152. T. Wakazawa et al., J. Antibiot. Ser. A 14

(1961) 180, 187.153. D. A. Preston, W. E. Wick, Antimicrob. Agents

Chemother. 1971, 322.154. M. J. Weinstein et al., Antimicrob. Agents

Chemother. (1961 – 70) 1964, 1.J. Black et al., Antimicrob. Agents Chemother.(1961 – 70) 1964, 138.

155. H. Umezawa et al., J. Antibiot. 24 (1971) 485.156. H. Kawaguchi, J. Infect. Dis. (Suppl.) 134

(1976) 242.157. R. Okachi et al., J. Antibiot. 27 (1974) 793.158. T. Shomura et al., J. Antibiot. 23 (1970) 155.159. S. A. Waksman, H. A. Lechevalier, Science

(Washington, D.C.) 109 (1949) 305.160. T. H. Haskell et al., J. Am. Chem. Soc. 81

(1959) 3480, 3482.161. A. Schatz, E. Bugie, S. A. Waksman, Proc.

Soc. Exp. Biol. Med. 55 (1944) 66.162. S. Kondo et al., J. Antibiot. Ser. A 18 (1965) 38.163. R. L. Mann, W. W. Broomer, J. Am. Chem.

Soc. 80 (1958) 2714.164. J. A. Waitz et al., Antimicrob. Agents

Chemother. 2 (1972) 431.C. C. Crowe, E. Sanders, Antimicrob. AgentsChemother. 3 (1973) 24.

165. S. A. Kabins et al., Antimicrob. AgentsChemother. 10 (1976) 139.J. J. Rahal et al., Antimicrob. AgentsChemother. 9 (1976) 595.V. Dhawan et al., Antimicrob. AgentsChemother. 11 (1977) 64.

166. D. J. Mason et al., Antibiot. Chemother.(Washington, D.C.) 11 (1961) 118.

167. R. Okachi et al., J. Antibiot. 30 (1977) 541.168. H. Umezawa et al., J. Antibiot. Ser. A 18

(1965) 101.169. J. M. McGuire et al., Antibiot. Chemother.

(Washington, D.C.) 2 (1952) 281.170. R. T. Bachand, J. Abtimicrob. Chemother. 27

(1991) 75.171. J.-F. Chantot, A. Bryskier, J.-C. Gasc, J.

Antibiot. 39 (1986) 660.172. C. H. Ballow, G. W. Amsden, M. D. Martinez,

M. Larouche, Ann. Pharmacother. 26 (1992)1253.

173. V. C. Stephens et al., J. Am. Pharm. Assoc. Sci.Ed. 48 (1959) 620.

174. T. Osono et al., J. Antibiot. 20 (1967) 174.K. Nitta et al., J. Antibiot. 20 (1967) 181.

175. J. C. Chabala et al., J. Med. Chem. 23 (1980)1134.

176. T. Tsuruoka et al., J. Antibiot. 24 (1971) 319,452.

177. W. Hausmann et al., J. Am. Chem. Soc. 76(1954) 4892.

178. B. A. Johnson et al., Science (Washington,D.C.) 102 (1945) 376.

179. Y. Koyama et al., J. Antibiot. 3 (1950) 457.180. M. H. McCormick et al., Antibiot. Annu. 1955

– 1956, 606.181. M. Rowland, Clincal Pharmacokinetics 18

(1990) 184.182. S. A. Waksman et al., Proc. Soc. Exp. Biol.

Med. 45 (1940) 609.183. W. Gold et al., Antibiot. Annu. 1955 – 1956,

579.184. E. L. Hazen, Science (Washington, D.C.) 112

(1950) 423.185. S. A. Waksman, Dtsch. Apoth. Ztg. 109 (1969)

1019.186. S. A. Goulden, Manuf. Chem. Aerosol News 36

(1965) no. 4, 45.187. C. T. Calan, Process Biochem. 7 (1972) no. 7,

29.188. American Cyanamid, GB 773453, 1954.189. Bristol-Myers, US 2970946, 1960.190. American Cyanamid, US 2734018, 1953 (P. P.

Minieri, H. Sokol, M. C. Firman).191. Bristol Lab., US 2739924, 1953.192. C. G. T. Evans, Manuf. Chem. 31 (1960) 5 – 9.193. G. D. Wilkin, Manuf. Chem. 31 (1960) 329.194. J. Malek, J. Hospodka, Folia Microbiol.

(Prague) 5 (1960) 120.195. J. Malek, Z. Fencl: Theoretical and

Methodological Basis of Continuous Culture ofMicroorganisms, Publ. House of the Czechosl.Acad. of Sciences, Prague, and AcademicPress, New York – London, Engl. ed. 1966.

196. A. L. Demain, C. L. Cooney, ProcessBiochem. 7 (1972) no. 7, 21.

197. W. Oberzill, N. Matsche, Chem. Ing. Tech. 43(1971) 83.

198. H. W. Blanch, P. L. Rogers, Biotechnol.Bioeng. 13 (1971) 843.

199. R. Kreutzfeldt, Angew. Chem. Int. Ed. Engl. 6(1967) 470.

200. M. J. Thirumalachar, R. S. Sukapure, P. W.Rahalkar, K. S. Gopalkrishnan, Hind. Antibiot.Bull. 5 (1962) 1.

201. B. N. Ganguli, V. M. Doctor, Hind. Antibiot.Bull. 7 (1964) no. 2, 85.

202. Distillers, GB 649818, 1948.203. Benckiser, DE 1000572, 1952.204. G. R. Ambekar, S. B. Thadani, Appl.

Microbiol. 13 (1965) 713.205. Ankerfarm, DE 1467764, 1965.

Antibiotics 55

206. Benckiser, DE 1103735, 1958.207. Hoechst, DE 1247549, 1965;

NL-A 6602132, 1966;BE 677053, 1966.

208. P. Hosler, M. J. Johnson, Ind. Eng. Chem. 45(1953) 871.

209. C. Rainbow, A. H. Rose: Biochemistry inIndustrial Microbiology, Academic Press,New York 1963, p. 254.

210. A. Soder: “Tetracycline,” in G. Ehrhart, H.Ruschig (eds.): Arzneimittel, Entwicklung,Wirkung, Darstellung, 2nd ed., vol. 4, VerlagChemie, Weinheim 1972, p. 368.

211. R. V. Reeves, Chem. Eng. (N.Y) 59 (1952)Jan., 145.

212. W. Durckheimer in [210] vol. 5, p. 302.213. Bundesverband der Pharmazeutischen

Industrie: “GMP-Regulations of Food andDrug Administration/USA,” 15. Jan. 1971,including amendment of 2. Mar. 1971, Pharm.Ind. 33 (1971) 364.

214. WHO: “Draft Requirements for GoodManufacturing Practice in the Manufacture andQuality Control of Drugs and PharmaceuticalSpecialities,” World Health Organisation,Tech. Rep., Ser. 1969, no. 418, Annex 2.

215. WHO: “Quality and Control of Drugs,”Official Records of the World HealthOrganisation, no. 176, Dec. 1972, Annex 12.

216. R. Marris, Pharm. Ind. 33 (1971) 749.217. Pharm. Ind. 32 (1970) 813 – 819.218. FDA, Fed. Regist. 37 (1972) no. 101, 10 510.219. E. P. Abraham, E. Chain et al., Lancet 1941,

vol. II, 177, 189.220. W. H. Schmidt et al., J. Bacteriol. 47 (1944)

199.221. M. D. Reeves et al., J. Bacteriol. 49 (1945)

395.222. FDA, Fed. Regist. 12 (1947), 4th Apr., 2215,

2217 – 2226.

223. H. Seyfarth, O. P. Ewald, Pharm. Ind. 34(1972) 40.

224. K. H. Wallhausser, Pharm. Ind. 34 (1972) 23.225. T. Dimmling, Arztl. Wochenschr. 8 (1953) no.

27, 633.226. P. Naumann, Antibiot. Chemother. (Basel) 10

(1962) 1 – 93.227. A. L. Barry, Am. J. Med. Technol. 30 (1964)

153, 333.228. L. J. Griffith, C. G. Mullins, Appl. Microbiol.

16 (1968) 656.229. L. G. Wayland, P. J. Weiss, J. Pharm. Sci. 57

(1968) 806.230. K. H. Wallhausser, Arztl. Lab. 16 (1970) 150.231. H. Knoll, Pharmazie 2 (1947) 392.232. W. Irmer, Dtsch. Med. Rundsch. 1949, 123.233. K. Irrgang, Z. Naturforsch. B: Anorg. Chem.

Org. Chem. Biochem. Biophys. Biol. 5 B(1950) 155.

234. G. Dorner, T. Lammers, Med. Klin. (Munich)46 (1951) 522.

235. W. H. C. Shaw, R. E. Duncombe, Analyst(London) 88 (1963) 694.

236. T. A. Haney et al., Ann. N.Y. Acad. Sci. 87(1960) 782;93 (1962) 627.

237. K. Heil, V. Beitz, Pharm. Ind. 34 (1972) 37.238. W. H. Shaw et al., Ann. N.Y. Acad. Sci. 130

(1965) 647.239. D. A. Burns et al., Biotechnol. Bioeng. 11

(1969) 1011.240. R. E. Hone, C. T. Rhodes, Process Biochem. 7

(1972) Feb., 27.241. E. P. Abraham, G. G. F. Newton, Adv.

Chemother. 2 (1966) 23.242. D. E. Nettleton et al., Int. J. Appl. Radiat. Isot.

13 (1962) 259.