Journal of Antimicrobial Chemotherapy (1977) 3 (Suppl. B), 29-39

In vitro studies with mecillinam on Escherichia coli and Pseudomonas aeruginosa

M. H. Richmond

Department of Bacteriology, University of Bristol, Bristol, BS8 1TD, England The ability of a 3-lactam antibiotic to inhibit the growth of Gram-negative bacteria depends on three main properties: the innate activity of the compound against cell wall synthesizing sites in the bacterial cell; the ability to penetrate through the outer layers of the bacterial envelope to these sites; and the ability to resist destruction by 3-lactamases that may be encountered on the way to the target. This article describes the properties of mecillinam with respect to the last two of these properties. Although able to hydrolyse mecillinam under some conditions, the 3-lactamases present in many Gram-negative species are unlikely to be very effective at protecting the bacteria in vivo because of their relatively low affinity for this penicillin and the good penetrative properties of the antibiotic. Introduction Experience over the last 25 years or so has shown that the clinical use of P-lactam antibiotics, at first penicillins alone, but latterly both penicillins and cephalosporins, has led to the emergence of resistant bacterial populations (Finland, 1971). Initially, the problem arose with strains of Staphylococcus aureus (Munch-Petersen & Boundy, 1962), but the advent of ampicillin as a widely used therapeutic agent has been followed by widespread resistance among Gram-negative species, notably Escherichia coli and other enteric strains (Medeiros, Kent & O'Brien, 1974). Nor has this process stopped even now. A year or so ago, we heard for the first time of the appearance of ampicillin resistant Haemophilus influenzae and of Haemophilus parainfluenzae strains (Medeiros & O'Brien, 1976) which caused grave clinical problems, and as recently as October 1976 the first reports of the isolation of gonococci resistant to high levels of penicillin have appeared in the medical literature (Phillips, 1976). So we find ourselves in the midst of a continually evolving situation, and one in which newly introduced penicillins and cephalosporins are likely to become blunted in their efficacy, even if they are fully effective when first used as therapeutic agents. In practice the resistance of Gram-negative bacteria to P-lactam antibiotics, if present, is based on two main processes: destruction of the antibiotic by degradative enzymes (p-lactamases in the case of P-lactam antibiotics) and exclusion of the antibiotics from their targets within the bacterial cells by an alteration in the properties of the bacterial cell envelope (Richmond & Curtis, 1974; Sykes & Matthew, 1976). Indeed, the overall properties of any penicillin or cephalosporin against Gram-negative bacteria may be 29

30

M. H. Richmond

evaluated against three aspects of their action:firstly,the inherent activity of the molecule against the mucopeptide (peptidoglycan) synthesizing systems in the 'inner membrane' of the bacterial cells; secondly, the resistance of the antibiotics to destruction by degradative enzymes; and thirdly, the ability of the antibiotic to penetrate (Richmond, Clark & Wotton, 1976). Once these three parameters of action have been measured one is on the way, at least, to predicting how the antibiotic will act. To date, progress has been rather unequal in investigating these three aspects of penicillin and cephalosporin action. The easiest of the three processes to quantitate is undoubtedly the response of the compound to the action of degradative enzymes (Sykes & Matthew, 1976). Penicillinases and cephalosporinases may be isolated in pure form from many Gram-negative species and their properties can be determined with accuracy (Richmond & Curtis, 1974; Sykes & Matthew, 1976). Studies on such preparations do not necessarily give a direct measure of how the enzymes act when present in intact bacteria (Hamilton-Miller, 1963), but nevertheless in vitro P-lactamase assays do certainly give important information about the therapeutic potential of a molecule. Of the remaining two aspects of P-lactam action, the activity of the molecules against mucopeptide (peptidoglycan) biosynthesis can be quantitated by measuring the activity of the molecule against isolated enzyme preparations (notably the transpeptidase) which acts at a late stage of cell wall biosynthesis in all bacteria (Nguyen-Disteche et al, 1974) Unfortunately, transpeptidase preparations are hard to make, and their substrates are complex and difficult to isolate. Nevertheless in vitro studies of the action of P-lactam antibiotics against transpeptidase preparations in vitro have been used to evaluate the innate effectiveness of some penicillins and cephalosporins against E. coli. The final aspect of penicillin and cephalosporin action is the ability of the compounds to penetrate the outer layers of the bacterial cells and to reach the site of mucopeptide biosynthesis. This aspect of penicillin and cephalosporin action has been the slowest to yield useful methods for its investigation, and it is only relatively recently that any studies directed towards the measurement of this aspect of the action of P-lactam antibiotics has been published. Basically the problem, as with all penetration studies in bacteria, is to decide what proportion of the uptake of a molecule is specific, and what proportion is directly related to the action of the antibiotic against its target. In practice, the difficulties of this type of measurement are so great that—with the exception of one or two recent sets of experiments (Blumberg & Strominger, 1974; Spratt, 1975)—indirect methods of assessing penetration have had to be used (Richmond & Curtis, 1974; Sykes & Matthew, 1976). Although some idea of the therapeutic potential of a new penicillin or cephalosporin may be obtained from studies of this kind, one should not assume that everything now falls neatly into place. Problems certainly still exist. The most difficult of these stems ultimately from the fact that the three basic aspects of P-lactam action—penetration, inhibitory effect and the ability to ward off the attentions of any P-lactamases that may be present—are interrelated and expressed in growing and multiplying bacteria. This means first that the P-lactamases are more effective at protecting bacterial cells when they are located behind a surface barrier which effectively impedes the passage of the antibiotic to the interior of the cell (Richmond & Curtis, 1974; Boman, Nordstrom & Normark, 1974). Secondly, the lethality of the inhibition of mucopeptide biosynthesis varies greatly on the rate of growth of the bacteria—something that has been well known from the earliest days of work with the penicillins (Hobby, Meyer & Chaffee, 1942). Finally, the external environment can play an important part: penicillins are much more

In vitro mecillinam on E. coli and Ps. aeraghwsa

31

effective in certain growth media than in others (Davis & Ianetta, 1972; D'Amato, Thornsberry, Baker & Kirven, 1975). Despite all these provisos, however, measurements on the stability of a new (3-lactam antibiotic to p-lactamases, on the ability of the molecules to penetrate, and on the innate activity of the molecule against transpeptidase systems all give valuable information for the evaluation of a new P-lactam antibiotic. In this article, we will be concerned primarily with mecillinam (FL1060). The studies reported in this paper are confined to some with isolated P-lactamases from Gram-negative species, and to an investigation of the molecule from the point of view of its penetrative properties. Studies on the intrinsic activity of the molecule against its target in the cell have been published elsewhere (Park & Burman, 1973; Matsuhashi et al., 1974). For a detailed discussion of the overall properties of this penicillin one should also look to the other contributions which make up this Symposium. Penicillin and cephalosporin (5-Iactamases Much work has been directed over the last 10 years or so to characterizing and classifying the P-lactamases to be found in Gram-negative bacteria of clinical importance (Richmond & Sykes, 1973; Richmond & Curtis, 1974). As implied by the term P-lactamase, all these enzymes open the P-lactam ring of the penicillin and/or cephalosporin nucleus to yield chemical compounds—sometimes complex mixtures of them that arise by further spontaneous chemical degradation of the primary product of enzyme action—which are antibiotically inactive. The enzymes are not, however, all equally active against all Plactam antibiotics. These differences in activity allow one to determine the substrate profile of a P-lactamase—the relative rate of activity of the enzyme against a range of widely available P-lactam compounds. In practice, if one uses substrate profile as an index, a range of different enzymes can be detected ranging from P-lactamases with a predominant activity against penicillins on the one hand, to an almost exclusively 'cephalosporinase' profile on the other (Jack & Richmond, 1970). Moreover, enzymes with the same profile are not always identical. Matthew and her collaborators at Glaxo have recently subjected a wide range of Plactamases to iso-electric focusing techniques (Sykes & Matthew, 1976) and has shown clearly that certain enzymes thought to be identical by substrate profile, and even sometimes on the basis of immunological examination as well (Sykes & Richmond, 1970), nevertheless separate differently when subjected to electrophoresis in the appropriate isoelectric focusing gradient. Initially, Richard Sykes and I (Richmond & Sykes, 1973) classified the P-lactamases we detected in Gram-negative species into five main classes among which we could discern 14 distinct enzyme types. This classification has subsequently been extended, and it is now possible to describe at least 25 different enzymes with a range of substrate profiles extending from one extreme of the range to the other (Sykes & Matthew, 1976). Furthermore, the addition of additional penicillins and cephalosporins to the clinical armoury usually results in the appearance of 'new' enzymes to be added to the classification. Against this highly complex background, it is clearly important to know which of these large range of enzymes are important clinically and which, in consequence, should be chosen for the in vitro evaluation of a new penicillin or cephalosporin. In the work to be described later in this article, we have used four enzyme preparations. Their properties and sources are set out in Table I. The first enzyme (Type la) has been chosen since it represents the most extreme 'cephalosporinase' profile that is likely to be encountered in

32

M. H. Richmond

clinically relevant strains (Richmond & Sykes, 1973). It is an enzyme found in species of Enterobacter cloacae (Goldner, Glass & Fleming, 1968; Ross, 1975) and has been implicated in a number of important clinical episodes of resistant infection. This enzyme is rarely of concern to those who seek to market a penicillin since these antibiotics are usually resistant to attack by this enzyme (Hennessey & Richmond, 1969). The second enzyme we have used—and probably the most important from the point of view of evaluating a new (3-lactam antibiotic, be it penicillin or cephalosporin—is the Type Ilia enzyme (Richmond & Sykes, 1973) (see Table I). It is an enzyme with a broad spectrum of activity which includes both penicillins and cephalosporins; but its most important character is that it is normally specified by a gene carried on a bacterial plasmid —or i?-factor (Datta & Richmond, 1966). This enzyme (sometimes called 'the TEM enzyme' from the i?-plasmid, RTEM, which specifies this enzyme and which was the first to be studied in this context) is widely (Datta & Kontomichalou, 1965) distributed through many Gram-negative species. It is, for example, responsible for the ampicillin resistance of the vast majority of resistant E. coli strains wherever they are detected, but the same enzyme is also responsible for the ampicillin resistance of the H. influenzae strains mentioned previously (Medeiros & O'Brien, 1976), and a very close relative of the Table I. Relative rates of hydrolysis by 0-lactamases together with their sources

(E. cloacae) PenG

100*

Amp

Carb

0 0

CER CEX CET CEZ

8000 1000 7200 3500

Cefoxitin Cefuroxime

Enzyme types Ilia (P. aeruginosa) (^-factor) Id

la

~0 0

IVc

(Aerobacter sp.)

100*

100*

100*

0 0 600 500 360 660 0 0

180 10 120 0-5 40 17 0 01

130 45 35 0-5 10 25 0 0-3

Abbreviations: Pen G, benzylpenicillin; Amp, ampicillin; Carb, carbenicillin; CER, cephaloridine; CEX, cephalexin; CET, cephalothin; CEZ, cefazolin. *A11 rates against Pen G adjusted to 100. Type Ilia enzyme was the cause of the carbenicillin resistance encountered in clinically serious circumstances among some strains of Pseudomonas aeruginosa a few years ago (Lowbury, Kidson, Lilly, Ayliffe & Jones, 1969; Sykes & Richmond, 1970). Recently this enzyme has also been implicated in the ampicillin-resistance of gonococci isolated in Liverpool (Phillips, 1976). We will be much concerned with this enzyme in relation to mecillinam—see below. The third enzyme we have used—Type IVc (see Table I) is also an enzyme with a broad spectrum encompassing both penicillins and cephalosporins (Richmond & Sykes, 1973). Unlike the Type Ilia enzyme, however, IVc enzyme will hydrolyse cloxacillin. This property is not, perhaps, of great clinical importance (unless one is contemplating the use of a mixture such as ampicillin/cloxacillin) but the difference in substrate profile reflects a very different type of P-lactamase. The enzyme is far less common in clinical strains than

In vitro mecillinam on E. eoli and Ps. aeruginosa

33

is the Type Ilia p-lactamase. Nevertheless, we feel that it presents a sufficiently different range of properties when compared with the other enzymes contained in this series, to make it worthwhile including it in the basic range of P-lactamases used for evaluation. The fourth and last enzyme type we have used is the Type Id enzyme found in all clinical isolates of Pseudomonas aeruginosa (Sabath, Jago & Abraham, 1965; Sykes & Richmond, 1971). It can be argued that many penicillins and cephalosporins are inactive against this bacterial species and therefore tests with this enzyme are superfluous. However, as we will see, penetration studies into Ps. aeruginosa may be very interesting from the point of developing new penicillins and cephalosporins; and these tests are difficult to evaluate unless one knows the response of the compound one is testing towards Type Id enzyme. This P-lactamase is primarily a cephalosporinase (like the Type la enzyme— see above) but retains more activity than that enzyme against some penicillins (Sabath et al., 1965). One can add in passing that any penicillin likely to be effective in treating Ps. aeruginosa infections must be resistant to this enzyme—a property notably present in carbenicillin (Sykes & Richmond, 1971). P-Lactamases and mecillinam Table II shows the relative stabilities of a number of penicillins and cephalosporins— including mecillinam—to the four P-lactamases included in this study. When assayed in this way mecillinam has very similar properties to ampicillin, that is the enzyme is susceptible to hydrolysis by Type Ilia and IVc enzyme, but not by Type la and Type Id. Table EL Relative rate of hydrolysis of mecillinam by P-lactamases Antibiotic

la

PenG Amp CER Mec

100* 0 8000 0

Enzyme type Ilia Id 100* 0 600 0

100* 180 120 23

IVc 100* 130 35 7

Abbreviations: Pen G, benzylpenicillin; Amp, ampicillin; CER, cephaloridine; Mec, mecillinam. •All rates against Pen G adjusted to 100.

The values in Table II were obtained by the iodometric method of P-lactamase assay (Novick, 1962) under conditions in which the substrate concentration was 10" SM at the beginning of the period of hydrolysis, and this amount of substrate is sufficient to saturate all the enzymes used in this test. However, such concentrations are much greater than are normally achieved in therapy, and it is important, therefore, to assess the stability of the compounds to p-lactamase at concentrations more typical of a therapeutic situation. To do this we need to assay the compounds at concentration of about 10" 5M, a procedure much more difficult than making the assays at the higher concentrations. In order to get over this problem, Pollock proposed that the 'physiological efficiency' of the P-lactamase should be measured. This quantity (defined as the Kmai value divided by the Km value) gives a measure of how effective the enzyme is likely to be at low substrate concentrations (Pollock, 1965). An example is given in Table III. The physiological

34

M. H. Richmond

Table HI. 'Physiological Efficiency' of Type n i a 3-lactamase and the effect of the enzyme on the resistance of E. coli to a number of P-lactam antibiotics Vm*i Km

E. coli MIC values (ng/ml) Resistant Sensitive

PenG Amp CER

7xlO» 7xlO» 5x10*

>500 >250 4

30 4 4

Mec

7x10*

8

8

Physiological efficiency: Va.JKa Abbreviations: Pen G, benzylpenicillin; Amp, ampicillin; CER, cephaloridine; Mec, mecillinam. efficiencies of Type Ilia enzyme are recorded in this table against cephaloridine and against ampicillin. The enzyme has an approximately equal hydrolysis potential against these two substrates (see Table I) but the Km values differ by about 50-fold. Thus ampicillin is much more effectively destroyed by the Type Ilia enzyme than is cephaloridine. When the physiological efficiency of Type Ilia enzyme is calculated for mecillinam a value about 100-fold lower than with ampicillin is obtained. Thus mecillinam is much more like cephaloridine than like ampicillin in its sensitivity to Type Ilia enzyme, and consequently it is only at high (non-therapeutic) concentrations that mecillinam is hydrolysed rapidly by Type Ilia enzyme. The physiological efficiency of the Type Ilia enzyme is related to its ability to protect p-lactamase-producing E. coli strains against p-lactam antibiotics. Table III also shows the typical MIC values obtained with P-lactamase-producing, and P-lactamase-nonproducing E. coli. The enzyme is much less effective at protecting lactamase-producing organisms against cephaloridine and against mecillinam than it is against ampicillin. Penetration of p-lactam antibiotics in E. coli It follows from the discussion in the previous sections of this contribution that the ability of a P-lactam antibiotic to pass through the outer layers of the envelope of Gramnegative bacteria plays a major role in the ability of that antibiotic to survive the attentions of any P-lactamase which happens to be present in the bacterial periplasmic space (see also Richmond & Curtis, 1974; Boman et al., \914 for a discussion of this point). How then are we to measure the penetrative ability of P-lactam antibiotics? One method that has been suggested is to measure the so-called 'crypticity' of the bacteria to the antibiotic in question (Hamilton-Miller, 1963; Richmond & Curtis, 1974). The basis of this method of measuring penetration is to argue that the ability of a Plactam antibiotic to reach the P-lactamase molecules in the periplasmic space is also a measure of the ease with which that compound can reach its target in the bacterial inner membrane. The structural basis of these relationships are summarized in Figure 1. In Y///////////VZO ™ IT"

ntp

°°,°°o °OO^° ]» im

Figure 1. Relative positions of p-lactamase molecules (o) and the target of penicillin and cephalosporin action (•) in the outer layers of E. coli cells. Abbreviations: om, outermembrane; mp, mucopeptide; ps, periplasmic space; im, inner membrane.

In vitro mecillinam on E. coli and Ps. aeruginosa

35

Table IV. Crypticity values for a number of P-lactam antibiotics measured with E. coli UB1005 (RPI) Crypticity PenG Amp

Carb CER

Mec

55 50 120 1-5 5

Abbreviations: Pen G, bsnzylpenicillin; Amp, ampicillin; Carb, carbenicillin; CER, cephaloridine, Mec, mecillinam.

practice 'crypticity' is determined by measuring the activity of a periplasmic P-lactamase when the bacteria are intact, and comparing the value obtained with that formed when the same number of bacteria are disrupted (by ultrasonic disintegration or some other means of cell breakage) and their enzymic activity measured (Richmond & Curtis, 1974). Typical values obtained with E. coli are shown in Table IV. Perhaps the most important generalization to come out of these studies is that penicillins frequently show large 'crypticity values' with E. coli, while cephalosporins show hardly any (Richmond & Curtis, 1974; Sykes & Matthew, 1976). The tentative conclusion from these studies is, therefore, that penicillins pass through the outerlayers of the E. coli envelope less freely than do the cephalosporins—a conclusion broadly supported by other methods of investigating antibiotic penetration. A substantial number of objections have been raised to the 'crypticity' method of determining penetration (Rosselet, Kniisel & Zimmermann, 1972; Richmond et al, 1976). Partly these are to do with the technical details of the measurement, but in principle the method cannot be used to measure the behaviour of P-lactam antibiotics that are insensitive to the P-lactamase in the periplasmic space (Richmond et al., 1976). In order to try to overcome these difficulties, we have recently developed an alternative method of assessing penetration (Richmond et al, 1976). It remains an indirect method,

0

10 20 30 40 50 60 Crypticity vs benzyl penicillin

Figure 2. Relative MIC values obtained with penicillins acting on E. coli UB1005 and on the mutants DC2 and DC3. Abbreviations for the penicillins used: A, ampicillin; G, benzylpenicillin; M, methicillin; C, cloxacillin. WT=strain UB1005. The crypticity values were obtained using E. coli UB1005(RPl). Data taken from Richmond et al. (1976) with the permission of the American Society for Microbiology.

M. H. Richmond

36

- 025 10 20 30 40 50 60 Crypticity vs benzyl penicillin

Figure 3. Relative MIC values obtained with cephalosporins acting on E. coli UB1005 and on the mutants DC2 and DC3. Abbreviations for the cephalosporins used: R, cephaloridine; X, cephalexin; T, cephalothin. WT=strain UB1005. The crypticity values were obtained using E. coli UB1005(RPl). Data taken from Richmond et al. (1976) with the permission of the American Society for Microbiology.

but it can be used for P-lactam antibiotics insensitive to P-lactamase hydrolysis. The method is based on the behaviour of E. coli mutants compared with a standard provided by the parental strain. The mutants have been selected by their inordinate sensitivity to penicillins, and this change in phenotype has been correlated with the extent of damage to their cell envelopes. In practice, two mutants have been used extensively (DC2 and DC3) and E. coli UB1005 has been used as the parental strain. Figure 2 shows how these strains may be used to assess the penetration of a P-lactam antibiotic (Richmond et al, 1976; Richmond & Wotton, 1976). The ordinate in this graph is the MIC of the strain in question against the antibiotic determined as a titration of single bacterial colony forming units on a series of agar plates containing a series of concentrations of the antibiotics in question. The abscissa is an arbitrary measurement of the degree of damage to the cell envelope—actually determined by a careful measurement of crypticity under highly standardized conditions. Figure 2 shows that cloxacillin is about 500 times more active against the mutant DC2 than against the parental strain. Thus this antibiotic seems to be powerfully excluded by the envelope of E. coli. Ampicillin shows an intermediate response being 60 times more active against DC2 than against 32 -

3 I -

- 8 WT~

DC 3

DC2 •Z

2

Me

E

o 2

• • - "

5 -

HO-5



012

o I

10

1

I

1

20

30

40

50

002

60

Crypticity vs benzyl penicillin

Figure 4. MIC values obtained with mecillinam (Me) acting on E. coli UB1005 and on the two mutant derivatives DC2 and DC3. WT=strain UB1005. The crypticity values were determined as described in Richmond et al. (1976).

In vitro mecillinam on E. coli and Ps. aeruginosa

37

UB1005, while, at the other extreme, cephaloridine (Figure 3) is almost as active against DC2 as it is with the parental E. coli (Richmond, Clark & Wotton, 1976). These results therefore confirm the earlier conclusions: penicillins tend to be excluded by E. coli, while cephalosporins are not. This method has also been used recently to assess the properties of a number of the newer cephalosporins (Richmond & Wotton, 1976). If we use this method to study mecillinam, we find a most interesting result (Figure 4). Unlike the majority of penicillins, mecillinam is almost as active against DC2 as against the parental E. coli UB1005. Thus, by this test, mecillinam seems markedly cephalosporinlike in its properties, at least inasmuch as cephaloridine itself is characteristic of that group of antibiotics. Penetration of mecillinam into Pseudomonas aeruginosa

Strains of Pseudomonas aeruginosa are unlike E. coli and other classical enteric species in that they show large 'crypticity' values both for penicillins and for cephalosporins (Richmond et ah, 1976); and this observation is confirmed when mutants of Ps. aeruginosa are used in the same way as DC2, DC3 and UB1005 (see above). With the pseudomonas mutants available to us at the moment, sensitivity to penicillins always goes hand in hand with sensitivity to cephalosporins, and the conclusion must be that both groups of P-lactam antibiotics have difficulty in getting into this bacterial species. Tests with mecillinam show that this antibiotic is no exception. With respect to wild-type Pseudomonas aeruginosa and two supersensitive mutants, mecillinam shows properties very similar to ampicillin. Conclusion

The studies reported here reinforce the view obtained from the work already published on mecillinam (see also this Symposium) that this antibiotic has many interesting properties. The studies already published on cell wall synthesis in E. coli suggest that this penicillin has very distinct properties from other p-lactam antibiotics (Park & Burman, 1973; Matsuhashi et al., 1974), and the work described here shows that many of the characteristics of the molecule—at least with respect to penetration and sensitivity to Type Ilia P-lactamase—are closer to those of cephaloridine than to ampicillin. Thus one might expect the molecule to have substantial activity against ampicillin-resistant strains of E. coli, bacteria that are becoming increasingly common (and worrying) in clinics throughout the world. Acknowledgements

I am grateful for grants from the Medical Research Council, and from Glaxo Research Ltd, to support some aspects of the original work reported here. Most of the antibiotics used in these studies have been the gifts of the appropriate British pharmaceutical companies. I also wish to thank the publishers of Antimicrobial Agents and Chemotherapy for permission to re-publish Figures 1 and 2 which had already appeared in their journal. References D'Amato, R. F., Thornsberry, C , Baker, C. N. & Kirven, L. A. Effect of Ca«+ and Mg!+ ions on the susceptibility of Pseudomonas species to tetracycline, gentamicin, polymixin B and carbenicillm. Antimicrobial Agents and Chemotherapy!: 596-600 (1975).

38

M. H. Richmond

Boman, H. G., Nordstrom, K. & Normark, S. Penicillin resistance in Escherichia coli K12: synergism between penicillinases and a barrier in the outer part of the envelope. Annals of the New York Academy of Sciences 235: 569-86 (1974). Blumberg, P. M. & Strominger, J. L. Interaction of penicillin with the bacterial cell: penicillin binding proteins and penicillin sensitive enzymes. Bacteriological Reviews3S:291-335(1974). Datta, N. & Kontomichalou, P. Penicillinase synthesis controlled by infectious /{-factors in Enterobacteriaceae. Nature (London) 208:239-41 (1965). Datta, N. & Richmond, M. H. The purification and properties of a penicillinase whose synthesis is mediated by an /{-factor in Escherichia coli. Biochemical Journal 98:204-9 (1966). Davis, S. D. & Ianetta, A. Influence of serum and calcium on the bactericidal activity of gentamicin and carbenicillin on Pseudomonas aeruginosa. Applied Microbiology 23: 775-9 (1972). Finland, M. Changes in susceptibility of selected pathogenic bacteria to widely used antibiotics. Annals of the New York Academy of Sciences 182:5-20 (1971). Goldner, M., Glass, D. G. & Fleming, P. C. Characteristics oiAerobacter P-lactamase. Canadian Journal of Microbiology 14:139-45 (1968). Hamilton-Miller, J. M. T. Penicillinase from Klebsiella aerogenes. Biochemical Journal 87: 209-14(1963). Hennessey, T. D. & Richmond, M. H. The purification and some properties of a p-lactamase (cephalosporinase) synthesised by Enterobacter cloacae. Biochemical Journal 109: 46973 (1969). Hobby, G. L., Meyer, K. & Chaffee, E. Observations on the mechanism of action of penicillin. Proceedings of the Society of Experimental Biology and Medicine 50:281-5 (1942). Jack, G. W. & Richmond, M. H. A comparative study of eight distinct 3-lactamases synthesised by Gram-negative bacteria. Journal of General Microbiology 61:43-61 (1970). Lowbury, E. J. L., Kidson, A., Lilly, H. A., Ayliffe, G. A. J. & Jones, R. J. Sensitivity of strains of Pseudomonas aeruginosa to antibiotics: emergence of strains highly resistant to carbenicillin. Lancet ii: 448-52 (1969). Matsuhashi, S., Tatsuyuki, K., Blumberg, P. M., Linnett, P., Willoughby, E. & Strominger, J. L. Mechanism of action and development of resistance of a new amidino penicillin. Journal of Bacteriology 117:578-87 (1974). Medeiros, A. A., Kent, R. L. & O'Brien, T. F. Characterisation and prevalence of the different mechanisms of resistance to beta-lactam antibiotics in clinical isolates of Escherichia coli. Antimicrobial Agents and Chemotherapy 6:791-800 (1974). Medeiros, A. A. & O'Brien, T. F. Ampicillin-resistant Haemophilus influenzae type B possessing a TEM-type 3-lactamase but little permeability barrier to ampicillin. Lancet i: 716—8 (1976). Munch-Petersen, C. & Boundy, E. Yearly incidence of penicillin resistant staphylococci in man since 1942. Bulletin of the World Health Organisation 26: 241-50 (1962). Nguyen-Disteche, M., Pollock, J. J., Ghuysen, J.-M., Puig, J., Reynolds, P., Perkins, H., Coyette, H. R. & Salton, M. R. J. Sensitivity to ampicillin and to cephalothin of enzymes involved in wall peptide cross-linking in Escherichia coli 44. European Journal of Biochemistry 41:457-63 (1974). Novick, R. P. The iodometric assay for penicillinase. Biochemical Journal 83: 236-40 (1962). Park, J. T. & Burman, L. FL1060, a new penicillin with a unique modeof action. Biochemical and Biophysical Research Communications 51:863-8 (1973). Phillips, I. P-lactamase-producing penicillin resistant gonococcus. Lancet ii: 656-7 (1976). Pollock, M. R. Purification and properties of penicillinases from two strains of Bacillus licheniformis: a chemical, physio-chemical and physiological comparison. Biochemical Journal^: 666-75 (1965). Richmond, M. H., Clark, D. C. & Wotton, S. Indirect method for assessing the penetration of beta-lactamase-nonsusceptible penicillins and cephalosporins in Escherichia coli strains. Antimicrobial Agents and Chemotherapy 10:215-8 (1976). Richmond, M. H. & Curtis, N. A. C. The interplay of beta-lactamases and intrinsic factors in the resistance of Gram-negative bacteria to penicillins and cephalosporins. Annals of the New York Academy of Sciences 235: 553-67(1974). Richmond, M. H. & Sykes, R. B. The P-lactamases of Gram-negative bacteria and their possible physiological role. In Advances in Microbial Physiology, Vol. 9 (Rose, A. H. & Tempest, D. W., Eds) Academic Press, London and New York (1973), pp. 38-88.

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Richmond, M. H. & Wotton, S. Comparative studies of seven cephalosporins: susceptibility to p-lactamases and the ability to penetrate the surface layers of Escherichia coli. Antimicrobial Agents and Chemotherapy 10: 219-22 (1976). Ross, G. W. (3-lactamase {Enterobacter species). In Methods in Enzymology, Vol. 43 (Hash, J. H., Ed.) Academic Press, London and New York (1975), pp. 678-86. Rosselet, A., Kniisel, F. & Zimmermann, W. Phenotypic expression of 3-lactamase activity in different strains carrying the TEM .R-factor. In Bacterial Plasmids and Antibiotic Resistance (Krcmery, V., Rosival, L. & Watanabe, T., Eds) Springer-Verlags Berlin, Heidelberg and New York (1972), pp. 231-43. Sabath, L. D., Jago, M. & Abraham, E. P. Cephalosporinase and penicillinase activities of a 3-lactamase fromPseudomonaspyocyanea. BiochemicalJournal96: 739-52 (1965). Spiatt, B. G. Distinct penicillin binding proteins involved in the division, elongation and shape of Escherichia coli K12. Proceedings of the National Accdamy of Sciences, USA 72: 29993003 (1975). Sykes, R. B. & Matthew, M. The P-lactamases of Gram-negative bacteria and their role in the resistance to P-lactam antibiotics. Journal of Antimicrobial Chemotherapy 2: 115-57 (1976). Sykes, R. B. & Richmond, M. H. Intergeneric transfer of a P-lactamase gene between Pseudomonas aeruginosa and Escherichia coli. Nature (London) 226:952-4 (1970). Sykes, R. B. & Richmond, M. H. .R-factors, 3-lactamase and carbenicillin-resistant Pseudomonas aeruginosa. Lancet ii: 342-4 (1971).

Note added in proof. In the course of this Symposium there has been frequent reference to the effect of high external solute concentrations on MIC values obtained with mecillinam acting on Gram-negative species. In my contribution to the Symposium, I described the use of some mutants of E. coli that could be used to evaluate the penetration of 3-lactam antibiotics to their targets in the bacteria] inner membrane. It, therefore, seemed interesting to test the effect of high solute concentration of the MIC values obtained with the mutants and with the parental culture. The figure (Figure 5) shows Figure 4 of the original paper redrawn with additional information added. It will be seen that the presence of high solute concentrations (10% w/v NaCl in this case) does indeed affect the MIC values obtained with mecillinam when the parental strain is used, but that this effect is not seen with the mutant DC2. This mutant is the one where the penetration barrier to the entry of many penicillins is missing (Richmond et al., 1976). These observations suggest tentatively, therefore, that the 'high solute effect' is exerted on the same features of the surface layers of E. coli that impede the entry of some penicillins (notably ampicillin) to its target in the E. coli inner membrane. 32 WT

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Figure 5. The MIC values obtained with mecillinam on a parental strain of E. coli and on two mutants, DC2 and DC3, which show reduced crypticity to benzylpenicillin and to ampicillin. This Figure is Figure 4 of the original contribution (see p. 36) redrawn to show the MIC's obtained when the strains are tested on nutrient agar (#) and on nutrient agar containing 10% (w/v) added NaCl (A). Crypticity of DC2 (2-5), crypticity of DC3 (20).

In vitro studies with mecillinam on Escherichia coli and Pseudomonas aeruginosa.

Journal of Antimicrobial Chemotherapy (1977) 3 (Suppl. B), 29-39 In vitro studies with mecillinam on Escherichia coli and Pseudomonas aeruginosa M...
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