Clinical and Epidemiologic Impact of Penicillins Old and New

Clinical and Epidemiologic Impact of Penicillins Old and New C. T. STEWART, M.D.

As therapeutic substances, penicillins gained new importance in 1959 with the discovery that their antibacterial and pharmacological usefulness could be widened by semisynthetic procedures involving the isolation of the common molecular nucleus 6-aminopenicillanic acid (6-APA) (Sakaguchi and Marao, 1950; Batchelor et aI., 1959), and the consequent practicability of varying side-chain structure. This discoverywhich came in a series of small steps, not accidentally as did the original discovery of penicillin-was applied in therapeutic practice in 1959 with remarkable despatch at a time when problems of antibiotic resistance and hospital-based infection due to staphylococci were showing signs of being almost insurmountable, so that the impact of discovery, for the second time in the history of penicillin, was obvious and immediate, as well as life-saving. This repetition of history and therapeutic importance has kept penicillin in the forefront of antibiotic development but only because, underlying these events, the antibiotic itself possesses unique properties, whose study amounts almost to a special branch of chemical biology. In this article, the medical uses of the penicillins are presented from this wider angle, since usage and conservation depend upon an understanding of the biologic origin and function of the penicillin molecule.

MOLECULAR STRUCTURE AND ACTIVITY

In structure, the penicillin molecule is deceptively simple (Fig. 1), being formed biosynthetically by fusion of the amino acids valine and cysteine to form a peculiar cyclic dipetide which gains antibacterial activity by virtue of an acyl side chain in the 6-position. The essential difference between the old and the new penicillins is that, in the old or natural penicillins, the acyl side chain is incorporated into the molecule by adding suitable precursors to the fermentation brew and letting the product form biosynthetically, whereas the new penicillins are formed Pediatric Clinics of North America-Vol. 15, No.1, February, 1968

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Nucleus (6-APA)

Side-chain

S . . - from cysteine

1

R· H2 CO - .

NHT-r rCH3)2-from "Ii" /""-

C - - N--CH·COO f + o

(Na or K salt or ester)

De cylase ex - arbon (absent in methicillin) ,a-lac amase Figure 1.

by adding a source of deacylating enzyme to a natural penicillin to release the molecular nucleus, 6-APA, which is then extracted from the brew and reacted with anyone of a wider range of side-chain precursors to give a semisynthetic product with desired biologic activity. It should be noted that, in general structure, the cephalosporin antibiotics are very similar to the penicillins, though the nucleus 7-aminocephalosporanic acid (7-ACA) has two loci available for substitution with potentially a wider range of biochemical reactivity which has not yet been realised. Chemically, the penicillins and cephalosporins form a large group of substances with a common ,8-lactam fused ring structure, which is the unique basis of their remarkably high, specific antibacterial potency. In accepting this potency as a kind of freakish bonus from nature, we tend to overlook the deeper ecologic significance of the ,8-lactam antibiotics. They are almost certainly peptide intermediates in the biosynthesis of a key generic protein in various moulds and fungi, including other species besides the type species from which the antibiotics are named. Since the fused ,8-lactam is unknown in forms of life lower than moulds, it can be postulated that, in evolution, the genetic variation that endowed a given primitive mould with the ability to form this kind of peptide conferred an immediate advantage for survival of that variant by giving it the power to eliminate bacterial competitors-presumably the only form of primitive life which would threaten the emerging moulds. The variant moulds would then thrive and diversify. Thus, penicillia, growing mainly on decaying organic substances and spreading by air, produced antibiotics which suppressed vegetative forms of airborne cocci and bacilli-a fact still exemplified by the therapeutic use of penicillin against these organisms and by the production, defensively, of penicillinases by staphylococci, Bacillus cereus and other organisms with which the penicillin-producing moulds have to compete

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in their natural habitat. The Cephalosporia, originally isolated from sewage and growing naturally in decaying organic liquids, produce antibiotics which direct themselves primarily against the gram-negative bacilli (coliforms, Aerobacter) which thrive in such liquids. In using the ,B-Iactam and certain other antibiotics therapeutically, we are intervening in an ecologic situation and altering a balance which nature cannot accept passively, as the emergence of various mechanisms of antibiotic resistance reveals. The intelligent use of antibiotics therefore requires some attention to biology as well as to therapeutic impulses.

MODE OF ACTION

The action of the ,B-Iactam antibiotics upon bacteria depends upon two properties of the bacteria themselves: (1) whether the organism has a cell wall in which a mucopeptide is the main component providing structural firmness; and (2) whether the organism can synthesize or learn to synthesize hydrolytic enzymes capable of breaking the carbonyl linkages in the ,B-Iactam molecule (Chain and Abraham, 1940). If a mucopeptide cell wall is present (condition 1) and specific hydrolytic enzymes (,B-Iactamases or acylases) are absent (condition 2), then the organism is highly vulnerable to attack by one or more of the ,B-Iactam antibiotics. In contrast, most other forms of life, including ~mall viruses, fungi (except actinomycetes), protozoa and practically all metazoan cells, are exempt from attack, since the relevant mucopeptides are absent and the enzyme irrelevant. Hence the nontoxicity of these antibiotics is part of their high antibacterial potency.

Resistance to the penicillins depends upon the same two factors. ( 1) The possession by bacteria of cell walls of different structure. This applies to many strains of Mycobacteria, streptobacilli, methicillinresistant staphylococci and some strains of Proteus and Pseudomonas. (2) Production of the inactivating enzymes ,B-Iactam acylase or amidase. The former enzyme is characteristic of resistant staphylococci (Kirby, 1945; Barber, 1947) and many gram-negative bacilli (Florey et al., 1949). Acylase or amidase are also found in coliforms, but their contribution to bacterial resistance is less certain (Hamilton-Miller, 1966). The rational usage of penicillins depends upon continuous recognition of these resistance factors, which change as bacterial populations adapt themselves to the antibiotic environment created by therapy. An additional mechanism of resistance, potentially of considerable clinical and epidemiologic importance though not yet fully assessed in this regard, is episomal resistance, which can be transferred by conjugation from one organism or species to another (Datta, 1965). The workings of these mechanisms are highly complex, but the main practical point which must be borne in mind if antibiotics are to retain their vital roles in prophylaxis and therapy is that administration of a given antibiotic, even to a single individual, constitutes a powerful selective pressure favouring, in the shorter or longer term, the emergence of resistant bacteria. The fact that penicillin G can still be used confidently against streptococci of group A and pneumococci is due to the absence of resistance mechanisms in these bacteria; but even while these

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bacteria are being eliminated, resistant forms of other species can be selectively . preserved.

Thus the widening use of penicillin G after 1945 favoured the replacement, in hospital and institutional environments, of penicillinsensitive staphylococci by highly resistant, penicillinase-forming staphylococci to such an extent that the usage of penicillin G against any form of infection, in hospital, began to be compromised. The penicillinasestable penicillins, especially methicillin, halted this deteriorating situation but only because, so far, alternative mechanisms of resistance are infrequent though, in some localities, by no means negligible (Chabbert, Baudens and Gerrand, 1965). The increasing use of broad-spectrum ,B-Iactam antibiotics (ampicillin, hetacillin, cephalosporins) is already leading to an increase in the prevalence of coliforms which produce inactivating enzymes, and it is possible that episomal or other mechanisms of resistance will be found useful by these bacteria. From the epidemiologic viewpoint, therefore, it is important to allow for the contingency that the genetic and biochemical adaptability of bacteria might be at least as inventive as our human capacity to produce new antibiotics.

THE OLD PENICILLINS

The discovery of natural penicillin by Fleming in 1929 and its rediscovery and isolation in a more or less pure state by Florey, Chain and Heatley, among others, at Oxford in 1940 are epics of medical history which lose none of their significance by recapitulation, though not in the present article. By 1945, the most stable and least toxic of numerous active penicillins, benzylpenicillin or penicillin G, was in widespread use, usually as the sodium or potassium salt in readily soluble, crystalline form which diffused rapidly, though for the most part unselectively, in most tissues after injection, and was excreted, 80 per cent unchanged, with equal rapidity. During the exciting period of Anglo-American collaboration (1941-45) which led to large-scale production of a relatively pure, inexpensive antibiotic, many attempts were made to synthesize and modify the molecule. Even at that time, synthesis was achieved on a small scale by condensation of D-penicillamine hydrochloride with 2benzyl-4-oxymethylene-5 ( 4) -oxazolone in the presence of triethylamine to form an intermediate which was then converted into the triethylammonium salt of benzylpenicillin (Du Vigneaud et aI., 1949). The yield, however, was too small and too costly to justify production by synthetic means. Biosynthesis by fermentative procedures therefore became the standard method of production of the drug, with consequences which were not apparent at the time and which are only now being fully realised. Largely because of the failure of synthetic production, modification of the molecule proved to be difficult and, in the early stages, did not reveal any useful changes in antibacterial spectrum or pharmacologic properties (Clarke, Johnson and Robinson, 1949). Esterification at the carboxyl group of benzylpenicillin gave a less soluble derivative, which paved the way, years later, for the introduction of the procaine salt of benzylpenicillin as a long-acting injectable compound. Emulsions in

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oils or waxes (Kirby et aI., 1945), notably an aluminum monostearate emulsion of procaine penicillin ( PAM), were used with more or less success to make depot preparations capable of releasing the active antibiotic for days or weeks after injection. A later derivative, benzathine penicillin (Bicillin) proved even more successful for this purpose and is now very widely used. The advantages of these long-acting preparations in prophylaxis of streptococcal sore throat, in rheumatic fever, in the treabnent of venereal disease and in various other circumstances are very real; the oft-quoted disadvantage of leaving a deposit of sensitising penicillin antigen in the tissues is admittedly a risk (Hsu and Evans, 1958; Steigman and Sliker, 1962) but, in the light of experience and newer knowledge of the mechanism of hypersensitivity (see below), a lesser risk compared with the risk of the infections which are undoubtedly prevented from developing. ' A further stimulus to modify the penicillin molecule followed observations, first recorded independently by various workers in 1944 (e.g., see Wilensky, 1946), that penicillin showed signs of becoming a major cause of local and systemic allergy. Logically enough, attempts were made to overcome this problem by modifying the side chain which was, in those days, added in the form of a suitable chemical precursor to the fermentation brew. Two derivatives prepared for this purpose, butylthiomethyl-penicillin and allylmercaptomethyl-penicillin, were found to be crossallergenic with benzylpenicillin (Risman and Boger, 1950).

During this period, the only derivative in which a substituted side chain brought a useful pharmacologic change was phenoxymethyl penicillin (penicillin V). This derivative had been prepared before 1945, but somehow its properties escaped even the attention of its originators (Behrens et aI., 1948) until Austrian workers (Brandl, Giovannini and Margreiter, 1953) showed that it was acid-stable and was therefore better absorbed than benzylpenicillin when given by mouth. This observation led to the realisation that acid stability could be conferred upon the molecule by substitution at the a-carbon, so that penicillin V, though produced fermentatively, was the forerunner of the semisynthetic oral penicillins. The price of acid-stability in this, though not at all a-substituents, was a tenfold loss in antibacterial power, but penicillin V was nevertheless sufficiently active to be used successfully in the prophylaxis and treatment of streptococcal and pneumococcal infections. The loss of activity was critical, however, in severe infections and some mishaps followed the use of penicillin V in syphilis, gonorrhoea and pyogenic meningitis-a fact which still has to be remembered in pediatric practice, as when meningococcal infection is abated but not cured by a few doses of penicillin V given to a child in the early stages of infection. Apart from penicillin V, penicillin G and its long-acting analogs, no derivatives of any therapeutic importance were produced until 1959, though several important biochemical advances were made during this period, as described in the paragraphs on molecular structure. In therapy, the penicillins were therefore used primarily-as predicted by Fleming in 1929-for infections caused by pyogenic cocci, for syphilis and, unexpectedly, for actinomycosis (Anderson and Keefer, 1948). They were often more useful than was realised in infections caused by certain gram-negative bacilli such as Escherichia coli and Proteus (Stewart, 1945; Thomas and Levine, 1945), a point of view which has been Vigorously upheld by Weinstein (1964, 1965). It became very quickly

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18 Table 1. Activity ORGANISM

of Penicillins Against Representative Pathogens

MINIMUM BACTERICIDAL CONCENTRATION OF MOST ACTIVE PENICILLIN

ALSO SUFFICIENTLY

(pg./ml.)

ACTIVE FOR THERAPY

Staph. aureus

Penicillin G 0.02-0.05

Staph. aureus (p'ase)

Cloxacillin *, nafcillin 0.2, methicillin 2.0 Pen. G, ampicillin 0.01-0.05

Strep. (group A) Strep. viridans Strep. group D Strep. (other groups) Pneumococcus Meningococcus Gonococcus Clos. welchii H. influenzae S. typhi Salmonella spp. Shigella spp. Proteus mirabilis t Proteus vulgaris t Esch. colit Pseudomonas

STEWART

Pen. V, phenethicillin, propicillin Oxacillin, dicloxacillin

Dicloxacillin, pen. V, phenethicillin, nafcillin Ampicillin 0.02-0.1, Pen G 0.02-10 Hetacillin, phenethicillin Ampicillin 1-5, Pen G 1-20 Hetacillin Hetacillin Ampicillin 0.02-5, Pen G 0.02-10 Hetacillin Pen. G, ampicillin 0.01-0.05 Pen. G, ampicillin 0.01-0.1 Pen. G, ampicillin 0.02-0.5 Pen. G, ampicillin 0.05-1 Ampicillin 0.01-5 Ampicillin 0.05-1 Ampicillin 2-5 Ampicillin 4 Ampicillin 4 Ampicillin 5-10 Ampicillin 2-5 Carbenicillin 6-200

* Other isoxazoles are slightly less active. t Penicillinase-forming strains are resistant.

apparent that the unique quality of natural penicillin was and is the combination of extremely high and relatively wide antibacterial activity combined with low, almost negligible toxicity. For the first time in history, and with the possible exception of water, every doctor and most witch-doctors everywhere had at their command a remedy with an undefined upper limit of dosage. It is impossible to estimate how many lives were saved and how much suffering was relieved by this fortunate coincidence, but there were some drawbacks-drug resistance, superinfection and allergy-which increased in proportion to the usage of penicillin and soon began to demand attention (Anderson and Keefer, 1948). The use and the limitations upon the use of penicillins today are to a surprising extent influenced by these facts, and that is why in this as in other important spheres of human affairs it is important in dealing with the present to remember the salient features of the past. The place of the older penicillins in prophylaxis and therapy today is narrower than it was prior to the discovery of newer antibiotics but still sufficiently important to make penicillin G the most widely used of all antibiotics (Table 1). With many other antibiotics available, however, it is necessary to keep evaluating its eligibility not only by laboratory assays of all organisms but also by comparing its pharmacologic properties with those of competitive antibiotics in relation to the known or

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presumed diagnosis. This latter category is especially important in the case of penicillin, which is often given while definitive diagnosis is pending.

THE NEW PENICILLINS

The availability in 1959 of 6-APA in bulk, first prepared at the Beecham Research Laboratories in England (Batchelor et aI., 1959) and developed industrially by that company and by Bristol Laboratories in the U.S.A., led rapidly to exploration of the changes in antibacterial and pharmacologic activity brought about by substitution with many different side chains. The new penicillins thus developed, and presently in use, can be classified as follows (Stewart, 1965): ( 1) Phenoxymethyl derivatives. In these, the a-carbon is substituted as in phenoxymethyl penicillin, giving acid-stability at the expense of lower activity and narrower antibacterial spectrum than that of penicillin G. This group includes phenethicillin, propicillin and phenpenicillin, of which only the first is used in the U.S.A. The therapeutic advantages of this group of drugs are marginal: they can be used instead of oral penicillin G in the prophylaxis of rheumatic fever and in the treatment of infections with pyogenic gram-positive cocci. If sufficiently high doses are given, phenethicillin can cure bacterial endocarditis due to Streptococcus viridans, which is usually inhibited by about 0.1 to 0.2 f-tg./ml. (Kennedy, Perkins and Jackson, 1962). In the presence of gastric hyperacidity, phenethicillin is a reliable form of oral penicillin but, in children, it is questionable if it possesses any advantage over penicillin G, which can be given orally in large doses more cheaply. (2) Derivatives with hindered side chains, usually in the form of bulky, electron-attracting substituents, which lower the susceptibility of substituted penicillins to the hydrolytic action of penicillinase. The mechanism of this interference is complex but probably depends mainly upon steric hindrance at the site of attachment of the enzyme. The result is that a variety of penicillins, substituted in this manner, are active against penicillinase-forming staphylococci. The prototype of this class, methicillin, was first used clinically to halt the epidemic spread of virulent penicillinase-forming staphylococci in hospitals in 1959 (Douthwaite and Trafford, 1960; Stewart et aI., 1960a, b) and might still be the penicillin of choice in the treatment of severe forms of infection with penicillinaseforming staphylococci (Hewitt, 1963; Barber and Garrod, 1963) because of its high stability to penicillinase and its safety when given parenterally in high doses. The intrinsic activity of methicillin is surprisingly low (about 1/100 that of pencillin G against staphylococci) but its bactericidal action is marked and a speedy, detoxifying therapeutic result is often observed in patients with systemic infections.

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Table 2. Semisynthetic Derivatives oj Penicillins in Therapeutic Use EFFECTIVE SIDE-CHAIN

DERIVATIVES

ROUTE

INDICATIONS

a-phenoxy-

Penicillin V Phenethicillin Propicillin *

Oral Oral Oral

Pneumococcal and streptococcal infections; prophylaxis ofrheumatic fever

Isoxazolyl-

Oxacillin Cloxacillin * Dicloxacillin *

Oral, 1M, IV Oral, 1M, IV Oral

P'ase staphylococci P'ase staphylococci P'ase staphylococci plus group A streptococci

Dimethoxybenzyl-

Methicillin

1M, IV

P'ase staphylococci

Ethoxynaphthamido-

Nafcillin

Oral, 1M

P'ase staphylococci, group A streptococci

a-aminobenzyl-

Ampicillin Hetacillin *

Oral, 1M, IV Oral

See Table 3

a-carboxybenzyl-

Carbenicillin * 1M, IV

Carboxyquinoxalinyl- Quinacillin *

Pseudomonas (Proteus, E. coli)

1M

* Available only for special investigation in U.S.A.

(December, 1967).

Besides methicillin, a number of other derivatives with hindered side-chains have been intensively studied in numerous reliable clinics. In most of these, the substituent involves the a-carbon of the side-chain, giving acid-stability. The most widely used of these derivatives are the isoxazolyl group and nafcillin, all of which can be given orally and are 10 to 20 times more active than methicillin, though more toxic and with a lower order of stability to penicillinase. Any of these drugs can be used with safety and confidence in the treatment of staphylococcal infections. Some of them are available in parenteral as well as oral formulation (Table 2), so that in more severe infections treatment can be initiated with intramuscular or intravenous dosage (Stewart, 1962b). In terms of intrinsic activity against penicillinase-forming staphylococci, cloxacillin is probably the best of the isoxazolyl penicillins (Stewart, 1962b; Kirby, 1964; Naumann, 1965). The activity of nafcillin is comparable, and this derivative is also widely used (Klein and Finland, 1963). It has to be remembered, however, that all these derivatives are, in general, less active than penicillin G or ampicillin against other pyogenic cocci and that, despite some claims to the contrary, they should not be used as a substitute for the more active penicillins unless the presence of a penicillinase-fOrming staphylococcus is known or suspected. In the isoxazolyl series, it is known that the addition of a chlorine atom to the phenyl ring of oxacillin (i.e., cloxacillin) increases activity slightly, and that the addition of two chlorine atoms (dicloxacillin) further increases activity selectively against streptococci of group A. This latter compound might therefore be specifically indicated when a mixed infection of ,B-streptococci and penicillinase-forming staphylococci is being treated, such as in impetigo and otitis media (Takasu, Babu and Kato, 1966).

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Because of the non toxicity of penicillin G and methicillin, there is a tendency to assume that each new penicillin can be used freely in large doses. The toxicity and allergenicity of penicillins is discussed in more detail below, and it may be said at this point that the isoxazolyl and naphthalene side chains of the derivatives mentioned are not free from toxicity. Doses in excess of 8 gm. per day carry distinct hazards in patients with any impairment of hepatic or renal excretion. The reason for this difference is that these more complex substances undergo metabolism in the liver and are therefore exc,reted more slowly, even in normal subjects. (3) Derivatives with additional functional groups. A functional group, in this context, means an atom or group of atoms conferring biochemical reactivity upon the molecule. The unsubstituted benzylpenicillin molecule is devoid of functional groups and acts on bacteria by structural interference. Before the development of the semisynthetic penicillins, it was apparent from work with precursors (Tosoni, Glass and Goldsmith, 1958) and penicillin N (Abraham and Newton, 1954) that the presence of a free amino group conferred extra activity against gram-negative bacilli. Of several substituted penicillins which utilise this fact, only two

Table 3.

Therapeutic Uses oj Ampicillin AMPICILLIN

ORGANISM

SENSITIVITY

RESISTANCE

INFECTIONS TREATED SUCCESSFULLY

0.01-0.05

Not reported

Wounds, impetigo, pyodermia, cellulitis Pneumonia, pharyngitis, otitis media

0.01-5.0

Very rare

Meningitis, endocarditis, peritonitis, septicemia

Hemophilus type B others

0.01-5.0 0.01-2.0

Exceptional Rare

Meningitis, septicemia Respiratory infections, chronic bronchitis

Pneumococcus (all types)

0.01-0.05

Not reported

Pneumonia, otitis media Endocarditis, meningitis

Meningococcus

0.01-0.1

(pg./ml.) Streptococcus group A

other groups

esp.

Not reported

Meningitis

E. coli

1-5

Common

Urinary, peritonitis, abscesses Pneumonia, meningitis

Proteus mirabilis

1-10

Common

Urinary, septicemia, wounds and burns Not recommended

other strains Salmonella typhi others Clostridia

Usually resistant

0.5 2-5 0.5-5.0

Not reported Rare

Typhoid fever and carriers Systemic salmonellosis

Rare

Wound, postpuerperal, systemic

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STEWART

are in regular therapeutic use, ampicillin and hetacillin. In each of these, substitution is at the a-carbon, so that oral therapy is practicable against pyogenic and other infections caused by sensitive gram-positive and gram-negative bacteria. Because of this, and because of its proven nontoxicity, ampicillin is an alternative or a replacement for the tetracyclines and chloramphenicol in situations where so-called broad-spectrum effects are desired. The place of hetacillin in therapy is very similar. Each of these penicillins is as active as and sometimes more active than penicillin G against streptococci, pneumococci and meningococci, and also inhibits most strains of Proteus mirabilis, Hemophilus and Salmonella. Many strains of E. coli are vulnerable, but penicillinase-forming strains of this organism, and of staphylococci, are resistant. Of all the new penicillins, ampicillin is now the most widely used, especially in pediatric practice, for the very good reason that extensive clinical experience has shown it to be safe and effective in a variety of infections, for initiating treatment in the absence of a bacteriologic diagnosis and, in many instances, for maintaining treatment thereafter. Because of its rapid bactericidal action upon many pathogens, it can be used, sometimes in preference to penicillin G, in serious infections. The usual dosage is 50 to 80 mg per kg. per day orally, but in meningitis, endocarditis and severe infections, and in typhoid carriers, dosage of 100 to 200 mg. per kg. per day is given intravenously. This can be maintained for weeks without toxicity, though skin rashes are common. Ampicillin is one of the few drugs-perhaps the only one-which will by itself control enterococcal endocarditis and salmonella osteitis, for which purposes doses in excess of 200 mg. per kg. per day are justified and are tolerated for 4 weeks or longer. The indications for hetacillin are similar, but clinical experience with this new derivative is much less extensive. The work of Wehrle and his colleagues at Los Angeles (Mathies et aI., 1966) suggests that ampicillin, given as bolus injections in a total dosage of 150 to 200 mg. per kg. per day into an intravenous infusion, might be the best primary drug in the treatment of pyogenic meningitis in children. The organisms most likely to cause meningitis in pediatric practice are meningococcus, hemophilus, pneumococcus, streptococcus and, in infants, E. coli and Pseudomonas. Ampicillin is as likely to be active against any of these organisms, except Pseudomonas, as any other single drug. The use of ampicillin therefore simplifies the urgent problem of immediate treatment while bacteriologic diagnosis is pending, and avoids the doubts and dangers of triple therapy. In the majority of their patients, the Los Angeles workers found that there was no need to alter therapy after bacteriologic diagnosis, and that the first results with ampicillin were as good as with any other form of therapy. In localised cerebral infections (e.g., after neurosurgery) with ventriculitis, ampicillin can be injected directly into the lateral ventricles (Stewart, 1965) since,

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like other penicillins, it penetrates into nervous tissue only in proportion to its plasma concentration in hyperemic zones. It has been reported recently (Acred et aI., 1967) that another simple derivative, carbenicillin, in which the a-carbon carries a carboxylic group, is active against Pseudomonas aeruginosa in concentrations of 6 to 200 fLg. per ml. This order of activity would be unremarkable except for the fact that, like other simple a-substituents, carbenicillin appears to be extremely low in toxicity, so that very large doses can be given. A clinical report (Brumfitt, Perival and Leigh, 1967) suggests that, in parenteral doses of 4 to 20 gm. daily, carbenicillin is effective in adults in controlling local, urinary and systemic infections with Pseudomonas aeruginosa. In a penicillin, activity against organisms in this group is highly unusual. Very occasionally, strains are inhibited by ampicillin at 50 fLg. per ml., but this drug will not arrest infections, which are, indeed, usually favored by administration of a penicillin. It remains to be seen if carbenicillin maintains its promise as mechanisms of resistance develop.

TOXICITY

Benzypenicillin and its simpler derivatives, including the a-phenoxy group, methicillin and ampicillin, are among the least toxic of all drugs. This is because, in the intact form, these penicillins are simple acylated peptides which are either excreted unchanged or degraded into simpler, equally nontoxic products. When reports about some so-called toxic effects (e.g., encephalopathies) due to penicillins are published (Brit. Med. J., 1967), the dosage is often stated in units, obscuring the fact that the load of electrolyte delivered with the antibiotic can in itself cause cardiovascular and neurologic effects. Dosage of 16 million units per day amounts to about 10 gm. of benzylpenicillin and 25 milliequivalents of potassium cation, which, delivered by continuous intravenous infusion, can certainly be toxic if cardiac or renal function are impaired, even temporarily. The sodium salt is not liable to cause cardiac embarrassment but it can provoke neuropathy if large doses are injected intravenously. These electrolytic problems are easily overcome by regulated injection and biochemical monitoring, so that doses of penicillin G, ampicillin and methicillin of 20 gm. per day or even more can be given in hospital to patients with endocarditis, osteomyelitis and pyemia, in which penicillin in one form or another is virtually irreplaceable. At this level of dosage, the antibiotics must be delivered by periodic (e.g., 3-hourly) injections delivered directly into an intravenous infusion. It has recently been found (Stewart, 1967a) that all these penicillins polymerise in solution. Hence all solutions for injection, whatever the dose, should be made up fresh, injected without delay and then discarded.

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With the oral penicillins, cation balance is regulated by alimentary absorption, and toxicity from this cause can be ignored. Problems of superinfection can arise with any form of oral penicillin by elimination of the competitive pharyngeal and intestinal flora, permitting overgrowth of resistant coliforms, Aerobacter, staphylococci, pathogenic yeasts (candida, cryptococci) or Aspergillus. Secondary syndromes including stomatitis, oesophagitis and enterocolitis can easily arise because of this effect but can more easily be prevented from developing by reasonable awareness of the possibility and by stopping or changing therapy when early signs of superinfection are detected. In this respect, the risks attending continuous oral therapy with penicillin are certainly no greater than those encountered with other antibiotics but, because of the rapidly changing pattern of microbial disease, especially in institutional environments, there is an increasing need to use antibiotics strategically and not speculatively. The absence of direct toxicity, characteristic of penicillin G, applies also to several derivatives including ampicillin, methicillin and phenethicillin. There is reason to believe, however, that the introduction of the more complex side chains used in the isoxazolyl derivatives and nafcillin can increase toxicity, due partly to the slower renal and hepatic excretion of these compounds and their metabolic products; in tissue cultures a direct toxic action lacking in the simpler derivatives is also seen with these compounds (Stewart, 1965). Even though the toxic effects so far observed with these compounds are attributable primarily to hepatic dysfunction (Medical Letter, 1962; Pas and Quinn, 1965), it is prudent to avoid giving doses in excess of 100 mg. per kg. per day for more than a few days unless it is certain that no other form of penicillin will serve the therapeutic purpose, which is an unlikely situation.

HYPERSENSITIVITY

It is generally agreed that hypersensitivity to penicillin is now one of the commonest forms of allergy. The prevalence varies surprisingly from 1 to 15 per cent in different clinics and according to different investigators but may be more reasonably estimated as 1 to 3 per cent of hospital or office patients. Estimates from positive reactions to skin test doses of penicillin G or penicilloyl polylysine (a suggested hapten model) give a higher prevalence of 5 to 10 per cent (Rytel et aI., 1963; Brown et aI., 1964), but of these, only a portion-50 per cent or less-actually react demonstrably to challenge with therapeutic doses (Smith, Johnson and Leighton, 1966). A much smaller proportion, less than 1 per cent of all reactors, give life-threatening anaphylactic reactions (Weinstein, 1965; Van Arsdel, 1965), usually to injections or tablets of natural penicillins

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in one form or another, in which the mortality rate is considered to be 9 to 13 per cent (Lancet, 1967). There is general agreement that the penicillins are all cross-reactive -that is, a patient known to be hypersensitive to penicillin G will react to any other form of penicillin (De Weck, 1962; Stewart, 1962) but not always to the same extent (Douthwaite et aI., 1961). Anaphylactic responses are seldom encountered except with the fermentatively prepared penicillins G and V. The explanation for this may lie in the recent finding (Batchelor et aI., 1967; Stewart, 1967b) that traces of a proteinaceous constituent of the fermentation brew persist in the final product even when purified according to standard U.S.P. specifications. This proteinaceous substance is only present in amounts of 10 to 30 mg. per 100 gm. of penicillin, but it carries the penicillin molecule, or part of it, as a hapten and is powerfully allergenic. A microgram or less will elicit allergic responses in sensitised subjects. A similar proteinaceous constituent is present in 6-APA prepared by deacylation but has not so far been detected in semisynthetic derivatives. This may explain why immediate anaphylactoid reactions occur with the natural penicillins and much less frequently, if at all, with the semisynthetic derivatives-which do give rise, quite frequently, to delayed reactions. Preliminary trials (Stewart, 1967b; Knudsen et aI., 1967) suggest that removal of the proteinaceous fraction lowers the allergenicity of penicillin G but may not overcome the problem of delayed reactions, in which other mechanisms are involved. Experimental studies (Levine, 1961; De Weck, 1962; Parker, 1963) have shown that penicillin itself, or its derivatives, cannot act as immunising or sensitising antigens unless they are linked as haptens to protein carriers. Being relatively unreactive biochemically, most penicillins are unable to form the necessary covalent bonds except by partial degradation. The hypothesis has therefore been advanced by Eisen, De Weck, Levine and Parker (Parker, 1963), as a result of extensive studies, that allergy is caused by multivalent antigens formed by linkages between penicillin products and tissue proteins. It has been further shown by these workers that the same products, used as univalent analogs, will inhibit some reactions, so it is possible that, in most individuals who receive penicillin, inhibitors neutralise the sensitising elements. Antibodies of yG and yM types are also formed (Watson et aI., 1961) and these may block the union of skin-sensitising or other reagenic (yE) antibodies with the penicilloylated protein antigens. Further proof that a mechanism of this nature is involved comes from the successful use, in a proportion of patients, of penicilloyl polylysine as a model of a degraded penicillin hapten conjugate, to detect hypersensitivity by skin tests (Parker, 1963), presumably by direct union with antibody at the site of injection. It is usually supposed that hypersensitivity to penicillins results from therapeutic use or misuse of these antibiotics, but in fact allergic and

G. T.

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STEWART

even anaphylactoid responses have been reliably recorded in individuals who have never received penicillins therapeutically, just as antibodies to penicillin-hapten complexes can be detected in similar persons. In these cases, sensitisation must have resulted from exposure to natural sources of penicillin-like substances produced by moulds and dermatophytes, or by penicillins present in milk and other foodstuffs, legally or otherwise (Siegel, 1959). The problem of causation is further complicated by recent evidence that natural and semisynthetic penicillins can polymerise by internal reactions to give macromolecules which, if aggregated with protein, might well form unusual antigens and explain some of the renal idiosyncrasies which have been occasionally reported with methicillin and isoxazolyl penicillins, at standard dosage. Even if the causes of allergy to penicillins are intricate and incompletely understood, the answers are relatively straightforward. If a patient has a history suggestive of allergy, penicillin should not be given if another antibiotic is equally suitable for treatment. On the other hand, if a penicillin is specifIcally required, preliminary skin tests with small doses (100 units or micrograms) of penicillin G or the appropriate derivative, and with penicilloyl polylysine if available, will detect true hypersensitivity in about 50 per cent of cases, and in these cases penicillins should be either avoided or used with great caution. In the remainder, penicillins can be given in incremental doses, starting with 1000 units or the equivalent weight. In all such situations, including skin tests, resuscitative measures (oxygen and a syringe loaded with epinephrine) should be at hand. In some instances, reactions can be "smothered" with antihistamines sufficiently for completion of a course of treatment (Green et aI., 1967). These measures may sound heroic but in many severe infections such as pyemia, endocarditis and osteomyelitis, penicillins are still the drugs of fIrst choice and the danger of allergy is often much less than the danger of withholding the drug.

GENERAL CONCLUSIONS

In range of antibacterial activity, pharmacologic properties and flexibility of dosage, the penicillins collectively have so many uses that it is easier to defIne contraindications, which are: (1) infections caused by certain gram-negative or gram-positive bacilli belonging to species which are invariably resistant-Aerobacter, Proteus morgani, Proteus rettgeri, Brucella, Pasteurella, Vibrio, Malleomyces, Mycobacteria; (2) infections caused by resistant strains of bacteria which are normally sensitive to one or more forms of penicillin, e.g., Proteus, E. coli, Staphylococci; (3) infections due to all Rickettsia, all Mycoplasma, all fungi except Actinomyces, all viruses except Psittacosis; and (4) The presence of specific allergy, provided an alternative form of therapy is practicable.

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The traditional place of penicillin G in the therapy of most streptococcal and pneumococcal infections, in gonorrhoea and in syphilis is in general unchanged though there are signs that in each of these ampicillin might be a rival with proven superiority in meningitis and some forms of streptococcal endocarditis. The availability of several forms of penicillinase-stable derivatives makes penicillin G and all other natural penicillins much less eligible for primary use in treating staphylococcal infections, against which methicillin and cloxacillin are of proven efficacy. Ampicillin and hetacillin extend activity to several gram-negative species, including Salmonella, and carbenicillin holds promise in infections due to Pseudomonas. Some of the severe reactions caused by the development of allergy appear to be preventible or avoidable as a result of the identification of removable macromolecular residues in penicillin G and in 6-APA. The improvement in the epidemiologic and therapeutic situation attributable to the new penicillins is threefold. In the first place, the penicillinase-stable derivatives are consistently effective against resistant staphylococci and there is no evidence as yet that the small minority of methicillin-resistant strains reported, mainly in Europe, are becoming more prevalent. Secondly, the range of antibacterial activity has been widened by ampicillin and its analogs and by carbenicillin. In some respects, ampicillin possesses higher intrinsic activity than penicillin G, and might become the penicillin of first choice if and when its price is lowered. Thirdly, several of the new preparations can be given orally as well as parenterally. Since toxicity is in general of a low order and since each new derivative is a fresh clue to the relationship between molecular structure and antibacterial activity, a new form of rational chemotherapeutic strategy is becoming practicable, based on molecular biology and microbial ecology. REFERENCES

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Levine, B. B.: Studies on the formation of the penicillin antigen. II. Some reactions of o-benzylpenicillenic acid in aqueous solution at pH 7.5. Arch. Biochem., 93:50, 1961. Mathies, A. W., Jr., Leedom, J. M., Thrupp, L. D., et al.: Experience with ampicillin in bacterial meningitis. Antimicrobial Agents and Chemotherapy 1966, p. 610. Medical Letter: Semi-synthetic penicillins. 4:29, 1962. Naumann, P.: Laboratory and clinical evaluation of cloxacillin. Antimicrobial Agents and Chemotherapy 1965, p. 937. Park, J. T. and Strommiger, J.: Mode of action of penicillin. Science, 125:99,1957. Parker, C. W.: Penicillin allergy. Amer. J. Med., 34:747, 1963. Pas, A. T., and Quinn, E. L.: Cholestatic ,hepatitis following the administration of sodium oxacillin. J.A.M.A., 191:674, 1965. Richmond, M. H.: Structural analogy and chemotherapeutic reactivity in the action of antimicrobial drugs. Symp. Soc. Gen. Microbiol., 16:301, 1966. Risman, G., and Boger, W. P.: Human skin sensitivity to penicillins G, BT, and 0, Demonstration of cross sensitization. J. Allergy, 21:425, 1950. Rytel, M. W., Klion, F. M., Arlander, T. R, and Miller, L. F.: Detection of penicillin hypersensitivity with penicilloyl polylysine. J.A.M.A., 186:844, 1963. Siegel, B. B.: Hidden contacts with penicillin. Bull. W.H.O., 21:703, 1959. Smith, J. W., Johnson, J. K, and Leighton, E. c.: Studies on the epidemiology of adverse drug reactions. New Eng. J. Med., 274:998, 1966. Sokaguchi, K., and Marao, S.: Preliminary report on a new enzyme "penicillinamidase". J. Agric. Chern. Soc. Japan, 33:411, 1950. Steigman, F., and Suker, J.: Fatal reactions to benzathine penicillin G. J.A.M.A., 288: 119, 1962. Stewart, G. T.: Effect of penicillin on BaciUus proteus. Lancet, 2:705, 1945. Stewart, G. T., Nixon, H. H., and Coles, H. M. T.: Report on clinical use of BRL 1241 in children with staphylococcal and streptococcal infections. Brit. Med. J., 2:703, 1960(a). Stewart, G. T., Harrison, P. M., and Holt, R J.: Microbiological studies on sodium 6- (2,6-dimethoxylbenzamido) penicillinate monohydrate (BRL 1241) in vitro and in patients. Brit. Med. J., 2:694, 1960(b). Stewart, G. T.: Cross-allergenicity of penicillins. Lancet, 1:509, 1962 a. Stewart, G. T., ed.: Report from six hospitals. Lancet, 2:634, 1962 b. Stewart, G. T.: The Penicillin Group of Drugs. Amsterdam, London and New York, Elsevier Publishing Co., 1965. Stewart, G. T.: Macromolecular residues associated with allergenicity in ,B-Iactam antibiotics. Antimicrobial Agents and Chemotherapy 1967, in press. Stewart, G. T.: Allergenic residues in penicillins. Lancet, 1:1177, 1967(b). Takasu, T., Babu, S., and Kato, J.: Clinical application of dicloxacillin to various infections. J. Antibiotics (B) Tokyo, 19(5):402, 1966. Thomas, A. R, Jr., and Levine, M,: Some effects of penicillin on intestinal bacteria. J. Bact., 49:623, 1945. Tosoni, A. L., Glass, D. G., and Goldsmith, L.: Crystalline p-aminobenzylpenicillin: Preparation and some properties. Biochem. J., 69:476, 1958. Van Arsdel, P. P.: Allergic reactions to penicillin. J.A.M.A., 191:238, 1965. Watson, K. C., Joubert, S. M., and Bennett, M. A.: Some factors influencing the hemagglutination of penicillin-sensitized erythrocytes. Immunol., 4: 193, 1961. Weinstein, L., Lerner, P. I., and Chew, W. H.: Clinical and bacteriologic studies of the effect of "massive" doses of penicillin G on infections caused by gram-negative bacilli. New Eng. J. Med., 271:525,1964. Weinstein, L.: Penicillin. In Goodman, L. S., and Gilman, A., eds.: The Pharmacological Basis of Therapeutics. New York, Macmillan, 1965, pp. 1193-1229. Wilensky, A. 0.: Fatal delayed anaphylactic shock after penicillin. J.A.M.A., 131:1384, 1946. School of Public Health University of North Carolina Chapel Hill, North Carolina 27514