The 50th anniversary of the discovery of 6-aminopenicillanic acid (6-APA).

This year (2007) marks the 50th anniversary of [corrected] discovery of 6-aminopenicillanic acid (6-APA), the precursor of all semi-synthetic penicillins [corrected] This review, by a scientist who played a major part in the discovery and a physician who participated in the early clinical trials of these antibiotics, tells the story of the discovery and of the early development of the beta-lactam antibiotics that revolutionised the treatment of infections.

Forty years of beta-lactam research.

For more than 40 years the author has been involved in research in the field of beta-lactam antibiotics. Much of this work was concerned with the development of the semisynthetic penicillins, following the isolation of the penicillin nucleus, 6-aminopenicillanic acid. This work resulted in the introduction of a number of important antibiotics including methicillin, cloxacillin, flucloxacillin, ampicillin, amoxycillin, carbenicillin and ticarcillin. Many of these compounds also provided the incentive or the basis for studies of a more fundamental nature in cell biology and antibacterial chemotherapy. The following is the author's personal account of some of the studies in the beta-lactam field in which he has been involved.

A review of the microbiology of amoxycillin/clavulanic acid over the 15 year period 1978-1993.

A review of the published literature over the period 1978-1993 was undertaken to assess any changes in the prevalence of beta-lactamase-producing bacterial pathogens and also any changes in the susceptibility in these pathogens to amoxycillin/clavulanate. The review has involved the examination of over 1500 publications. The review shows that in general there has been an increase in the frequency of beta-lactamase-producing pathogens resistant to ampicillin and amoxycillin but there is no evidence for any significant increase in resistance to amoxycillin/clavulanate over the period of the review.

Evolution of beta-lactamase inhibitors.

The production of beta-lactamase is the most important mechanism of bacterial resistance to beta-lactam antibiotics. Attempts to find an inhibitor of beta-lactamase were made as early as the 1940s and 1950s but without success. In the early 1950s, it was found that certain semisynthetic penicillins could function as beta-lactamase inhibitors, but none found a clinical place in this capacity. A program of screening microorganisms for the production of naturally occurring inhibitors was begun in 1967. This process led to the discovery of the olivanic acids and clavulanic acid. Clavulanic acid, formulated with amoxicillin and later with ticarcillin, became available for clinical use in 1981. Since the introduction of clavulanic acid, other beta-lactamase inhibitors have been developed, including sulbactam and tazobactam. It remains to be seen whether these will have any advantage over clavulanate for clinical use.

Evolution of beta-lactamase inhibitors.

In most clinical isolates that are resistant to penicillins, cephalosporins and related compounds, the mechanism of resistance is the production of beta-lactamase enzymes. These enzymes hydrolyze the amide bond in the beta-lactam ring of the compound, producing acidic derivatives that have no antibacterial properties. The rationale for beta-lactamase inhibitors is to overcome this resistance. Early study of beta-lactamase inhibition was begun in the 1940s without success. Interest in beta-lactamase inhibition was renewed with the development of the semisynthetic penicillins in the early 1960s, when it was found that certain of these compounds could function as inhibitors. However, none found clinical application. The screening of microorganisms for possible production of naturally occurring beta-lactamase inhibitors resulted in the discovery of the olivanic acids and, later, clavulanic acid. A formulation of clavulanic acid with amoxicillin was introduced in 1981, and a formulation of clavulanic acid with ticarcillin appeared shortly thereafter. More recently, other beta-lactamase inhibitors have been developed, including sulbactam and tazobactam. A formulation of sulbactam with ampicillin has appeared recently. As a result of beta-lactamase inhibition, amoxicillin and clavulanate and ticarcillin and clavulanate are active against a high proportion of amoxicillin resistant and ticarcillin resistant pathogens. These formulations have been shown to be safe and effective in the treatment of many infections that would not be expected to respond to amoxicillin or ticarcillin alone.

Evolution of beta-lactamase inhibitors.

Beta-lactamase, the bacterial enzyme that can inactivate penicillins, cephalosporins and related antibiotics, can function outside the cell or in the periplasmic space. This resistance can be transferred between bacteria of the same or different species. Most strains of Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella, Enterobacter, Pseudomonas aeruginosa and Bacteroides species are beta-lactamase producers. Clavulanic acid is a beta-lactamase inhibitor that works by blocking the enzyme center. When it is combined with amoxicillin and ticarcillin, it expands those drugs' spectrum of activity to bacteria that are resistant to the single antibiotics as well as to anaerobic bacteria.

Sensitivity of clinical isolates from German hospitals to amoxicillin/clavulanic acid (Augmentin) compared with other antibiotics.

17,244 pathogens isolated from clinical specimens of 24 hospitals in the Moers area (North-Rhine Westphalia, FRG) were tested in regard to their susceptibility to Augmentin (amoxicillin and clavulanic acid). For this purpose, minimal inhibitory concentrations were determined by use of microbroth dilution technique. 80% of Gram-negative, 98% of Gram-positive and 97% of anaerobic isolates were susceptible to Augmentin (breakpoint 4 mg/l amoxicillin in the presence of 2.5 mg/l clavulanic acid). In a second part of the study the susceptibility to Augmentin of 4.137 Gram-negative and 10.958 Gram-positive pathogens was compared to their sensitivity against benzylpenicillin, flucloxacillin, mezlocillin, erythromycin, clindamycin, fusidic acid, ampicillin, cefaclor and doxycyclin.

Tissue penetration of antibiotics.

A novel method of deduction is described for determining the amount of antibiotic present in the tissues at any point in time following intravenous administration of a given dose. Application of this calculation to amoxycillin, carbenicillin, ticarcillin, nafcillin, dicloxacillin and temocillin indicates that the passage of drug from the vascular compartment to the extravascular fluid is rapid. The results obtained with this method of calculation are discussed in relation to the results obtained by conventional pharmacokinetic analysis and by direct assay of tissue fluid samples.

Factors affecting the apparent regrowth of Pseudomonas aeruginosa following exposure to bactericidal concentrations of carbenicillin.

Factors have been identified which are responsible for the phenomenon already reported in which regrowth of Pseudomonas aeruginosa was found to occur in broth cultures containing bactericidal concentrations of carbenicillin. In cultures incubated under stationary conditions, in a water bath, the factor primarily responsible for this phenomenon of regrowth appears to be the formation of condensate on the inside of the culture vessel. In such condensate, viable cells of P. aeruginosa were found in numbers equal or higher than those in the culture broth. As a result of coalescing, and running down the vessel wall, the condensate provides a continuous reintroduction of bacteria into the culture medium, and, notwithstanding a bactericidal concentration of antibiotic in the medium, this process of continual reinoculation leads to the formation of visible growth and an increase in the viable count in the culture medium after an initial bactericidal effect. In silicone-treated flasks, incubated in a water bath, regrowth did not occur. Under such conditions, condensate again formed, but only as discrete droplets on a non-wetting surface, and in such condensate the bacterial count was found to be low. In flasks incubated in an air incubator, condensate did not form, and under these conditions the phenomenon of regrowth was not observed.

Selection of variants of Gram-negative bacteria with elevated production of type 1 beta-lactamase.

Variants of Pseudomonas aeruginosa, Citrobacter freundii and Enterobacter cloacae were isolated that produced elevated levels of type 1 beta-lactamase. The variants were readily isolated on antibiotic-containing agar and showed resistance to a wide range of beta-lactam antibiotics including some regarded as being relatively stable to the beta-lactamase produced.

Effect of protein binding on antibiotic activity in vivo.

Using a group of penicillins all belonging to the same chemical class, antibacterial activity against Staphylococcus aureus was determined in vitro and also in vivo by use of an intraperitoneal infection in mice. The compounds all showed essentially the same level of activity in vitro but differed markedly in their activity in vivo. This activity in vivo could be correlated directly with the extent of binding in mouse serum.

The history and background of Augmentin.

Bacterial resistance to the beta-lactam group of antibiotics is frequently due to the production of beta-lactamase which brings about the inactivation of the antibiotic. Clavulanic acid is a naturally occurring inhibitor of beta-lactamase which is capable of rendering penicillin- and cephalosporin-resistant organisms sensitive. The compound is obtained by fermentation from Streptomyces clavuligerus. Clavulanic acid shows some structural similarity to the penicillins and cephalosporins and functions as a progressive inhibitor of a wide range of beta-lactamases including those found in Escherichia coli, Klebsiella aerogenes, Proteus species, Bacteroides fragilis, Haemophilus influenzae, Neisseria gonorrhoeae and Staphylococcus aureus. Clavulanic acid is well absorbed when given by mouth and a formulation with amoxycillin (Augmentin; Beechams) is now available for clinical use.

Regrowth of Pseudomonas aeruginosa and other bacteria after the bactericidal action of carbenicillin and other beta-lactam antibiotics.

Exposure of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus to bactericidal concentrations of beta-lactam antibiotics in broth culture resulted in a decrease in viability over the first 6--8 hr, followed by regrowth which was not due to the selection of resistant variants or loss of antibiotic potency. During incubation, bacteria adhered to the surface of the culture vessel and multiplied despite the presence of bactericidal concentrations of antibiotic in the medium. It is concluded that the phenomenon of "regrowth" results from such adhesion and the subsequent dispersal of some of these cells into the culture medium. The significance of these findings is discussed in relation to the treatment of infection, the determination of minimal bactericidal concentrations, and the phenomena of tolerance and persisters.

Effect of beta-lactam antibiotics on bacterial cell growth rate.

The effect of penicillins and cephalosporins on the rate of growth of individual cells of Escherichia coli on agar was determined by measurement of cell length using photomicrographs taken at frequent intervals. When exposed to cephalexin or carbenicillin at twice the minimum inhibitory concentration cell division was inhibited but growth in length proceeded at a uniform exponential rate until abruptly terminated by lysis. The rate of growth of such filamentous cells did not differ significantly from that of normal cells grown in the absence of antibiotic. In contrast, exposure to cephaloridine and amoxycillin at twice the minimum inhibitory concentration resulted in a marked diminution in the rate of cell growth, and lysis occurred very much sooner. At concentrations greatly in excess of the minimum inhibitory concentration (greater than 100-fold) cephalexin also resulted in a diminution in the rate of cell growth and this was accompanied by earlier lysis. The significance of these findings is discussed in relation to the mode of action of beta-lactam antibiotics.