Journal of Antimicrobial Chemotherapy (1977) 3 (Suppl. B), 5-11
Antibacterial activity of mecillinam
D. S. Reeves
Southmead Hospital, Bristol, England
When Lund and Tybring first described the properties of mecillinam in 1972 they pointed out that this compound differed from known P-lactam antibiotics in two main respects, (i) Mecillinam was highly active against certain Gram-negative bacteria but had much reduced activity against Gram-positive bacteria, unlike penicillins and cephalosporins where the reverse is usually the case, (ii) Mecillinam appeared to cause microscopical changes in cultures of Escherichia coli which were hitherto undescribed and which differed from those caused by penicillins and cephalosporins. The first of these properties, that of high activity of mecillinam against Gram-negative organisms, excited interest because of its potential value in therapy, and therefore the activity of mecillinam against pathogenic bacteria will be reviewed, together with factors affecting this activity and its measurement, and synergistic activity with other antibacterial drugs. Mode of action Light microscopy studies The earliest intimation of the unique mode of action of mecillinam was in light microscopy studies (Lund & Tybring, 1972; Melchior, Blom, Tybring & Birch-Andersen, 1973). Cultures of E. coli treated with ampicillin or benzylpenicillin (called penicillin hereafter for convenience) at low concentrations (around the IC50) form filamentous cells. On raising the concentration the addition of an osmotic stabiliser is necessary or lysis will occur. At concentrations slightly above the IC 60 small protusions appear on the rod shaped cells. At 5 to 10 times the IC 5 0 'rabbits' ears' cells occur before the final appearance of a predominance of spheroplasts. The morphological response to mecillinam does not vary with concentrations from the ICSO to a 1000 times greater and consists of formation initially of ellipsoidal cells and then large spherical cells, with lysis of the remaining cells. This change is not dependant on the presence of an osmotic stabiliser. With a stabiliser a typical penicillin type response (i.e. 'rabbits' ears') is seen at very high concentrations of mecillinam (50,000 XIC B0 ). Division slowly continues, the spheroidal cells having a very extended doubling time ( x 3 control). Eventually after some hours the spheroidal cells lyse without first forming spheroplasts. Cell death from mecillinam also occurs slowly at very low concentrations without lysis (Matsuhashi et ah, 1974).
Downloaded from http://jac.oxfordjournals.org/ at Monash University on June 19, 2015
Introduction
6
D. S. Reeves
A similarity to penicillin type effects is that division must continue during the period of action of mecillinam if lysis is to occur. Thus mecillinam activity can be prevented by chloramphenicol or nalidixic acid (Matsuhashi et al, 1974), or by treating a culture of high viable count nearing the stationary phase (Tybring & Melchior, 1975). An interesting corollary to the dramatic changes in cell size and shape induced by mecillinam is that optical density cannot easily be used to follow the effect of the drug on viable count in fluid cultures.
Cell-wall synthesis Low concentrations of mecillinam have been shown to inhibit the synthesis of murein by 50 to 70 % in both growing cultures of E. coli and in static bacteria, but there was no increase in the inhibition by using increasing concentrations of mecillinam (Park & Burman, 1973). Ampicillin caused a reduction of murein synthesis which progressed nearly to completion on increasing the concentration. Unlike ampicillin, mecillinam had no effect on the synthesis of cross-linked murein. Mecillinam had no effect on the activity of two enzymes known to be normally inhibited by penicillin, endopeptidase and D-alanine carboxypeptidase. Nevertheless, the effect of mecillinam on murein synthesis and on cell morphology indicates that its ultimate mode of action must be on cell wall metabolism. Recently, Spratt & Pardee (1975) have shown that mecillinam competed with high affinity against penicillin for a minor penicillin binding protein in E. coli inner membrane. (The binding only accounted for 0-5 % of all penicillin binding which perhaps explains why the phenomenon was not discovered before using less sensitive techniques.) 6-Aminopenicillanic acid, the only other P-lactam antibiotic so far shown to produce 'ovoids' at its lowest effective concentration, also displayed high affinity for the same binding protein as mecillinam. Furthermore, a mutant E. coli which failed to show binding of penicillin to this protein grew in culture as round cells. Penicillin also binds to the same protein component, but the effect is presumably masked by its more severe effects on peptidoglycan transpeptidases. Activity of mecillinam against pathogenic bacteria The initial reports by Lund & Tybring (1972; Table I) of high activity against Gramnegative bacteria and much reduced activity against Gram-positive organisms have been
Downloaded from http://jac.oxfordjournals.org/ at Monash University on June 19, 2015
Electron microscopy These show diffuse surface changes with no special predeliction for the division site (Greenwood & O'Grady, 1973a). The surface shows irregular pitting. This, coupled with the continued ability to divide suggests that there must be at least two sites of action for p-lactam antibiotics, one being concerned with preventing division and the other with preventing generalized mucopeptide synthesis. Greenwood & O'Grady (19736) have pointed out that cephalexin, the action of which is predominantly to produce filaments, in combination with mecillinam produces a 'typical' penicillin response. Electronmicrographs of ultra-thin sections of E. coll show the cell walls of bacteria treated with mecillinam to be very similar to those from control cultures (Melchior et al., 1973). If anything, the intermediate layer shows slight thickening. In contrast, the cell walls of bacteria from osmotically stabilized cultures treated with penicillin, show the intermediate layer to be thin or nearly absent.
Antibacterial activity of mecillinam Table I. Activity of mecillinam against individual strains. (Values taken from Lund & Tybring, 1973) IQo mg/1 Ampicillin
Penicillin
0015 010 010 016 010 160 50 100 0-5 016 16
016 100 0-50 100 10 500 016 50 0 006 0005 016
050 100 1-6 100 3-2 1000 0008 50 0005 0004 050
confirmed by others. Our results (Reeves, Wise & Bywater, 1975; Table II) give rather higher values, perhaps reflecting differences in technique and in reading. Other workers (Greenwood, Linton Brooks, Gargan & O'Grady, 1974; McGhie, Robertson, Geddes & Lim, 1976) have, like us, found some cross-resistance between ampicillin and mecillinam, although this is not in keeping with the results of Tybring (1975). The activity of mecillinam in vitro depends greatly on the conditions under which it is studied, perhaps more than with other p-lactam antibiotics, and a review of some of these factors is therefore appropriate. Inoculum size Changes in inoculum size have greater effects on mecillinam activity than on other P-lactam antibiotics even if P-lactamase-producing strains are excluded. For example, we found increasing the inoculum from 1 x 103 to 1 x 107 could increase the MIC by more than 100-fold, although it was variable (Table III; Reeves, Wise & Bywater, 1975). Large decreases in activity were almost invariable with Proteus mirabilis and Klebsiella spp., but much less common with E. coli, Salmonella spp. and Enterobacter cloacae. Strains which were resistant to ampicillin tended to show a large inoculum effect, presumably because they were P-lactamase producers. But there are a number of reasons why, apart
Table II. Activity of mecillinam—typical values. (Reeves, Wise & Bywater, 1975) MIC mg/1 E. coli (ampicillin sensitive) E. coli (ampicillin resistant) Klebsiella spp. Proteus mirabilis Proteus spp. (Indole+VE) Salmonella spp. Staph. aureus (penicillin sensitive) Strep, faecalis
0-25 1 00 10 25 >50 0-25 50 50
Downloaded from http://jac.oxfordjournals.org/ at Monash University on June 19, 2015
Esch. coli Klebsiella pneumoniae Proteus mirabilis Enterobacter cloacae Salmonella typhimurium Pseudomonas aeruginosa Staph. aureus (penicillin sensitive) Staph. aureus (penicillin resistant) Strep, pyogenes Neisseria gonorrhoeae H. influenzae
Mecillinam
8
D. S. Reeves
Table m . Effect of inoculum size on mecillinam activity. (Unpublished data. Reeves, Wise & Bywater)
Organism (strain)
Mecillinam 103 10'
E.coliM E. coli 102 Proteus mirabilis 145 Klebsiella spp.
Antibacterial activity of mecillinam
D. S. Reeves
Southmead Hospital, Bristol, England
When Lund and Tybring first described the properties of mecillinam in 1972 they pointed out that this compound differed from known P-lactam antibiotics in two main respects, (i) Mecillinam was highly active against certain Gram-negative bacteria but had much reduced activity against Gram-positive bacteria, unlike penicillins and cephalosporins where the reverse is usually the case, (ii) Mecillinam appeared to cause microscopical changes in cultures of Escherichia coli which were hitherto undescribed and which differed from those caused by penicillins and cephalosporins. The first of these properties, that of high activity of mecillinam against Gram-negative organisms, excited interest because of its potential value in therapy, and therefore the activity of mecillinam against pathogenic bacteria will be reviewed, together with factors affecting this activity and its measurement, and synergistic activity with other antibacterial drugs. Mode of action Light microscopy studies The earliest intimation of the unique mode of action of mecillinam was in light microscopy studies (Lund & Tybring, 1972; Melchior, Blom, Tybring & Birch-Andersen, 1973). Cultures of E. coli treated with ampicillin or benzylpenicillin (called penicillin hereafter for convenience) at low concentrations (around the IC50) form filamentous cells. On raising the concentration the addition of an osmotic stabiliser is necessary or lysis will occur. At concentrations slightly above the IC 60 small protusions appear on the rod shaped cells. At 5 to 10 times the IC 5 0 'rabbits' ears' cells occur before the final appearance of a predominance of spheroplasts. The morphological response to mecillinam does not vary with concentrations from the ICSO to a 1000 times greater and consists of formation initially of ellipsoidal cells and then large spherical cells, with lysis of the remaining cells. This change is not dependant on the presence of an osmotic stabiliser. With a stabiliser a typical penicillin type response (i.e. 'rabbits' ears') is seen at very high concentrations of mecillinam (50,000 XIC B0 ). Division slowly continues, the spheroidal cells having a very extended doubling time ( x 3 control). Eventually after some hours the spheroidal cells lyse without first forming spheroplasts. Cell death from mecillinam also occurs slowly at very low concentrations without lysis (Matsuhashi et ah, 1974).
Downloaded from http://jac.oxfordjournals.org/ at Monash University on June 19, 2015
Introduction
6
D. S. Reeves
A similarity to penicillin type effects is that division must continue during the period of action of mecillinam if lysis is to occur. Thus mecillinam activity can be prevented by chloramphenicol or nalidixic acid (Matsuhashi et al, 1974), or by treating a culture of high viable count nearing the stationary phase (Tybring & Melchior, 1975). An interesting corollary to the dramatic changes in cell size and shape induced by mecillinam is that optical density cannot easily be used to follow the effect of the drug on viable count in fluid cultures.
Cell-wall synthesis Low concentrations of mecillinam have been shown to inhibit the synthesis of murein by 50 to 70 % in both growing cultures of E. coli and in static bacteria, but there was no increase in the inhibition by using increasing concentrations of mecillinam (Park & Burman, 1973). Ampicillin caused a reduction of murein synthesis which progressed nearly to completion on increasing the concentration. Unlike ampicillin, mecillinam had no effect on the synthesis of cross-linked murein. Mecillinam had no effect on the activity of two enzymes known to be normally inhibited by penicillin, endopeptidase and D-alanine carboxypeptidase. Nevertheless, the effect of mecillinam on murein synthesis and on cell morphology indicates that its ultimate mode of action must be on cell wall metabolism. Recently, Spratt & Pardee (1975) have shown that mecillinam competed with high affinity against penicillin for a minor penicillin binding protein in E. coli inner membrane. (The binding only accounted for 0-5 % of all penicillin binding which perhaps explains why the phenomenon was not discovered before using less sensitive techniques.) 6-Aminopenicillanic acid, the only other P-lactam antibiotic so far shown to produce 'ovoids' at its lowest effective concentration, also displayed high affinity for the same binding protein as mecillinam. Furthermore, a mutant E. coli which failed to show binding of penicillin to this protein grew in culture as round cells. Penicillin also binds to the same protein component, but the effect is presumably masked by its more severe effects on peptidoglycan transpeptidases. Activity of mecillinam against pathogenic bacteria The initial reports by Lund & Tybring (1972; Table I) of high activity against Gramnegative bacteria and much reduced activity against Gram-positive organisms have been
Downloaded from http://jac.oxfordjournals.org/ at Monash University on June 19, 2015
Electron microscopy These show diffuse surface changes with no special predeliction for the division site (Greenwood & O'Grady, 1973a). The surface shows irregular pitting. This, coupled with the continued ability to divide suggests that there must be at least two sites of action for p-lactam antibiotics, one being concerned with preventing division and the other with preventing generalized mucopeptide synthesis. Greenwood & O'Grady (19736) have pointed out that cephalexin, the action of which is predominantly to produce filaments, in combination with mecillinam produces a 'typical' penicillin response. Electronmicrographs of ultra-thin sections of E. coll show the cell walls of bacteria treated with mecillinam to be very similar to those from control cultures (Melchior et al., 1973). If anything, the intermediate layer shows slight thickening. In contrast, the cell walls of bacteria from osmotically stabilized cultures treated with penicillin, show the intermediate layer to be thin or nearly absent.
Antibacterial activity of mecillinam Table I. Activity of mecillinam against individual strains. (Values taken from Lund & Tybring, 1973) IQo mg/1 Ampicillin
Penicillin
0015 010 010 016 010 160 50 100 0-5 016 16
016 100 0-50 100 10 500 016 50 0 006 0005 016
050 100 1-6 100 3-2 1000 0008 50 0005 0004 050
confirmed by others. Our results (Reeves, Wise & Bywater, 1975; Table II) give rather higher values, perhaps reflecting differences in technique and in reading. Other workers (Greenwood, Linton Brooks, Gargan & O'Grady, 1974; McGhie, Robertson, Geddes & Lim, 1976) have, like us, found some cross-resistance between ampicillin and mecillinam, although this is not in keeping with the results of Tybring (1975). The activity of mecillinam in vitro depends greatly on the conditions under which it is studied, perhaps more than with other p-lactam antibiotics, and a review of some of these factors is therefore appropriate. Inoculum size Changes in inoculum size have greater effects on mecillinam activity than on other P-lactam antibiotics even if P-lactamase-producing strains are excluded. For example, we found increasing the inoculum from 1 x 103 to 1 x 107 could increase the MIC by more than 100-fold, although it was variable (Table III; Reeves, Wise & Bywater, 1975). Large decreases in activity were almost invariable with Proteus mirabilis and Klebsiella spp., but much less common with E. coli, Salmonella spp. and Enterobacter cloacae. Strains which were resistant to ampicillin tended to show a large inoculum effect, presumably because they were P-lactamase producers. But there are a number of reasons why, apart
Table II. Activity of mecillinam—typical values. (Reeves, Wise & Bywater, 1975) MIC mg/1 E. coli (ampicillin sensitive) E. coli (ampicillin resistant) Klebsiella spp. Proteus mirabilis Proteus spp. (Indole+VE) Salmonella spp. Staph. aureus (penicillin sensitive) Strep, faecalis
0-25 1 00 10 25 >50 0-25 50 50
Downloaded from http://jac.oxfordjournals.org/ at Monash University on June 19, 2015
Esch. coli Klebsiella pneumoniae Proteus mirabilis Enterobacter cloacae Salmonella typhimurium Pseudomonas aeruginosa Staph. aureus (penicillin sensitive) Staph. aureus (penicillin resistant) Strep, pyogenes Neisseria gonorrhoeae H. influenzae
Mecillinam
8
D. S. Reeves
Table m . Effect of inoculum size on mecillinam activity. (Unpublished data. Reeves, Wise & Bywater)
Organism (strain)
Mecillinam 103 10'
E.coliM E. coli 102 Proteus mirabilis 145 Klebsiella spp.
