Journal of Antimicrobial Chemotherapy (1975) 1, 97-101
The antibacterial activity of a new cephalosporin, cefamandole
A. D. Russell
The antibacterial activity of a new cephalosporin, cefamandole, has been assessed. The antibiotic was active against high and low inocula of penicillin-sensitive and P-lactamase-producing strains of Staphylococcus aureus and against large inocula of various strains of Proteus sp. (especially Pr. mirabilis, although Pr. vulgaris appeared to be resistant) and non p-lactamase-producing Escherichia coli. Concentrations much higher than minimum inhibitory levels were needed to bring about lysis. Typical penicillin-like morphological variants were induced in Gramnegative bacteria.
Introduction Cefamandole is a new water-soluble cephalosporin derivative. Chemically, it is the sodium salt of 7-D-mandeIamido-3-{l-methyl-lH-tetrazol-5-ylthiomethyl)-3-cephem-4carboxylic acid (Figure 1). Cefamandole has been reported to have a broad spectrum of activity, including activity against Haemophilus strains and indole-positive and indolenegative strains of Proteus (Wick & Preston, 1972). The present report describes the inhibitory levels of cefamandole on some benzylpenicillin-sensitive and resistant strains of Staphylococcus aureus, on various Proteus strains and other Gram-negative bacteria, including carbenicillin-sensitive and R + strains of Pseudomonas aeruginosa and potent P-Iactamase producers in Escherichia coli R+TEM, Enterobacter cloacae P99 and Klebsiella aerogenes K l (Marshall, Ross, Ghanter & Harris, 1972). In addition, the effects of cefamandole on growth, lysis and induction of morphological variants in some strains have been studied. Materials and methods Bacterial strains These consisted of penicillin-susceptible and ^-lactamase producing strains of S. aureus and various strains of Ps. aeruginosa and other Gram-negative bacteria. All strains were grown overnight in nutrient broth (Oxoid) at 37°C. Minimum inhibitory concentrations (MICs) MIC values were determined by placing 0-05 ml of a 1 in 100 dilution (c. 5 X 10' viable cells/ml) of an overnight culture of an organism on to the surface of overdried agar 97
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Welsh School of Pharmacy, University of Wales Institute of Science and Technology, Cathays Park, Cardiff, Wales
98
A. D. Russell
plates containing the desired cefamandole concentration. The plates were incubated at 37°C for 24 h and examined for the presence or absence of growth. The minimum concentration completely preventing growth was taken as the MIC.
N
COOH
N
N N I CH 3
Figure 1. Chemical structure of cefamandole.
Results and discussion The MIC values of cefamandole against various strains of S. aureus and of Gramnegative bacteria are shown in Tables I and II. Although only a limited number of strains has been tested the following conclusions may be tentatively reached: (a) cefamandole is active against penicillin-sensitive and ^-lactamase producing strains of S. aureus. Inoculum size appears to have only a slight effect, if any; (b) non beta-lactamase producing strains of E, coli (NCTC 9001, NCTC 100418 and R~TEM) are sensitive to cefamandole, whereas the only p-lactamase producer tested (E. coli R+TEM) is highly resistant; (c) other Gram-negative bacteria which are powerful producers of j5-lactamase (Enterobact. cloacae P99 and K. aerogenes Kl) are also highly resistant to cefamandole (cf. the non-fi-lactamase-producing mutant, Enterobact. cloacae P99M, in Table II); (d) all strains of Ps. aeruginosa tested, irrespective of whether or not they are carbenicillin sensitive or possess R-factors, are highly resistant to cefamandole; (e) all strains of Pr. mirabilis and some strains of Pr. rettgeri and Pr. morganii are sensitive to cefamandole, whereas the strains of Pr. vulgaris are resistant. Cephaloridine shows the same pattern of response with the strains listed in (b) and (c) above (Russell, 1972), but is particularly prone to changes in inoculum size with staphylococci under (a) (Ridley & Phillips, 1965; Benner, Bennett, Broadie & Kirby, 1965). Cephaloridine is also active against most strains of Pr. mirabilis (Eykyn, 1971), and thus the two antibiotics show a similar response in this context The effects of adding cefamandole to growing cultures of various strains are shown in Figures 2 to 7. The antibiotic induces lysis of P99M (Figure 2) and R~TEM (Figure 3)
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Effect of cefamandole on rate of growth 0-1 ml vol. of overnight 37°C broth cultures were added to 9-4 ml of nutrient broth in nephelos flasks (Pyrex flasks with attached side-arms) previously equilibrated at 37°C in a shaking incubator (100 oscillations/min). When the cultures had entered the logarithmic phase of growth at 37°C, 0-5 ml of differing cefamandole solutions were added to give the desired final drug concentration. Incubation was continued at 37°C. Turbidity changes before and after the addition of the antibiotic were monitored with the EEL Unigalvo. In cases where the turbidity of cultures continued to increase after the addition of cefamandole, samples were removed for microscopical examination (phase contrast microscope, x 900). Photographs of these samples were taken by means of a Kodak camera attached to a Vickers microscope.
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Plate 1. Morphological changes in some Gram-negative bacteria exposed to cefamandole at stated concentration, (a) Pr. mirabilis 10374, 20 ng/ml, 250min; (b)Pr. mirabilis 10374, 10 ng/ml, 250 min; (c)Pr. vulgaris4M5, 20tig/ml, 250min; (d) Pr. rettgeri 8893, 20 ng/ml, 270min; (e)Pr. rettgeri8893, 10(ig/ml, 270 min; (0 Pr. morganii 235, 20 tig/ml, 250 min; (g) E. co// R"TEM, lOjig/ml, 300 min; (h) Enterobact. cloacae P99M, 20 ng/ml, 300 min; (i) P99M, 10 ng/ml, 300 min.
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Antimicrobial activity of a new cephalosporin, cefamandole
99
Table I. MIC values for cefamandole against some strains of staphylococci MIC of cefamandole (ng/ml) Organism S. aureus
Oxford NCTC 3761 1605* 3452* 11127* 1613*
A
B
C
0-5 0-5 5 0-5 0-5 10
5 0-5 0-5 2-5
2-5 0-5 0-5 2-5
but a concentration of 20 ng/ml, or about 4 to 8 times the respective MIC, is needed before this is achieved. Concentrations of up to 20 |xg/ml were also tested with other strains; as would be expected from the MIC values in Table n , 20 ji-g/ml cefamandole had a negligible effect on the growth of R + T E M and P99. However, cefamandole at 20 p-g/ml did not cause lysis or markedly reduce the growth rate of Pr. mirabilis NCTC 10374 (Figure 4) or of the highly ampicillin-sensitive Pr. morganii NCTC 235 (Figure 5); some lysis of Pr. rettgeri NCTC 8893 occurred at 5 and 10 (xg/ml, followed by re-growth, with a rather more extensive lysis at 20 ng/ml (Figure 6). Surprisingly, cefamandole at 10 and especially 20 ng/ml, reduced the rate of growth of Pr. vulgaris NCTC 4175, but no lysis occurred (Figure 7) and, at 24-h incubation, extensive growth of this organism had occurred in all concentrations. Samples were removed from those flasks which continued to show an increase in Table n . MIC values for cefamandole against some strains of Gram-negative bacteria Strain
Organism Pr. vulgaris
Pr. morganii Pr. rettgeri
NCTC 10015 NCTC 4175 NCTC 10376 J. 27* NCTC 235 NCTC 1707 NCTC 5845 NCTC 8893 NCTC 7475 NCTC 10371 J23* H* H2*
Pr. mirabilis
NCTC 6369 NCTC 10374 NCTC 9589 S4*
S39* S33*
MIC ((ig/ml) 100 100
>100 100 7-5 5 100 2-5 10 100 7-5 15 50 7-5
Organism
Strain
Ps. aeruginosa R+3425 R+1822 NCTC 6750 NCTC 8203 NCTC 10701 NCTC 10490 NCTC 1999 E. coli NCTC 9001 NCTC 100418 R-TEM R+TEM Enterobact cloacae
P99 P99M
MIC (ng/ml) >1000 >1000 >1000 >1000 >1000 >1000 >1000 2-5 1 1 500 500 5
2-5-5 5 25 2-5 2-5
Kl. aerogenes Kl
* Hospital isolates. CEM cefamandole.
>500
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* ()-lactamase producers. A, B, C, . . . 1 in 100, 1 in 10,000 and 1 in 1,000,000 dilutions,respectivelyof an overnight culture.
A. D. Russell 100
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Figures 2-7
2
0 Tlmeot37#C(h)
6
Antimicrobial activity of a new cephalosporin, cefamandole
101
Acknowledgements The author thanks Dr C. F. Speirs of Lilly Research Ltd, Windlesham for generous supplies of cefamandole; Kodak Ltd, for financial help with the photomicrographic studies; and Mrs J. A. Harris, Miss L. M. Smart and Mr D. Collins for technical assistance. References Benner, E. J., Bennett, J. V., Broadie, J. L. & Kirby, W. M. M. Inactivation of cephalothin and cephaloridine by Staphylococcus aureus. Journal of Bacteriology 90: 1599-1604 (1965). Eykyn, S. Use and control of cephalosporins. Journal of Clinical Pathology 24: 419-29 (1971). Eykyn, S., Jenkins, C , King, A. & Phillips, I. Antibacterial activity of cefamandole, a new cephalosporin antibiotic, compared with that of cephaloridine, cephalothin and cephalexin. Antimicrobial Agents and Chemotherapy 3: 657-61. Marshall, M. J., Ross, G. W., Ghanter, K. V. & Harris, A. M. Comparison of the substrate specifities of the P-lactamase from Klebsiella aerogenes 1082E and Enterobacter cloacae P99. Applied Microbiology 23: 765-9 (1972). Ridley, M. & Phillips, I. Relative instability of cephaloridine to Staphylococcal penicillinase. Nature, London 208: 1076-8 (1965). Russell, A. D. In vitro studies with CIBA 36,278-Ba, a new cephalosporin derivative. Microbios 6: 221-30. (1972). Russell, A. D. Effect of some p-lactam antibiotics on the morphology of some P-lactamase and non-P-lactamase producing strains of Gram-negative bacteria. Journal of Applied Bacteriology 36: 357-9 (1973). Wick, W. E. & Preston, D. A. Biological properties of three 3-heterocyclic-thiomethylcephalosporin antibiotics. Antimicrobial Agents for Chemotherapy 1: 221-34 (1972). [Manuscript accepted 19 November 1974)
Figure 2. Effect of cefamandole (added at 4 ) on the growth of E. coli R"TEM in broth at 37°C. Cefamandole concentrations (ng/ml): 0, A—A; 2-5, • — • ; 5, o—O; 10, • — • ; 20, D—D. Figure 3. Effect of cefamandole (added at | ) on the growth of Enterobact. cloacae P99M in broth at 37°C. Symbols: as in Figure 2. Figure 4. Effect of cefamandole (added at j ) on the growth of Pr. mirabilis N.C.T.C. 10374 in broth at 37°C. Symbols as in Figure 2. Figure 5. Effect of cefamandole (added at j ) on the growth of Pr. morganil N.C.T.C. 235 in broth at 37°C. Symbols as in Figure 2. Figure 6. Effect of cefamandole (added at | ) on the growth of Pr. rettgeri N.C.T.C. 8893 in broth at 37°C. Symbols as in Figure 2. Figure 7. Effect of cefamandole (added at | ) on the growth of Pr. vulgaris N.C.T.C. 4175 in broth at 37°C. S>mbols as in Figure 2.
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turbidity and examined by phase-contrast microscopy. Examples of the morphological forms induced by cefamandole are shown in Plate 1. These indicate that increases in turbidity may be the result of morphological variants, which are typical of those induced by p-lactam antibiotics in p-lactamase and non-p-lactamase producing Gram-negative bacteria (Russell, 1973). It must be pointed out that much higher numbers of viable bacteria are of necessity present in these growth experiments than in the MIC experiments described in Tables I and II. Thus, bearing this in mind, it may be concluded (i) that inoculum size may be of importance with these Gram-negative strains, (ii) that the minimum lytic concentration, which would correspond to a minimum bactericidal concentration (MBC) with a [J-lactam antibiotic, is several times higher than the MIC. This latter point confirms the recent finding made by Eykyn, Jenkins, King & Phillips (1973) for cefamandole and a wider range of Gram-negative bacteria.
The antibacterial activity of a new cephalosporin, cefamandole
A. D. Russell
The antibacterial activity of a new cephalosporin, cefamandole, has been assessed. The antibiotic was active against high and low inocula of penicillin-sensitive and P-lactamase-producing strains of Staphylococcus aureus and against large inocula of various strains of Proteus sp. (especially Pr. mirabilis, although Pr. vulgaris appeared to be resistant) and non p-lactamase-producing Escherichia coli. Concentrations much higher than minimum inhibitory levels were needed to bring about lysis. Typical penicillin-like morphological variants were induced in Gramnegative bacteria.
Introduction Cefamandole is a new water-soluble cephalosporin derivative. Chemically, it is the sodium salt of 7-D-mandeIamido-3-{l-methyl-lH-tetrazol-5-ylthiomethyl)-3-cephem-4carboxylic acid (Figure 1). Cefamandole has been reported to have a broad spectrum of activity, including activity against Haemophilus strains and indole-positive and indolenegative strains of Proteus (Wick & Preston, 1972). The present report describes the inhibitory levels of cefamandole on some benzylpenicillin-sensitive and resistant strains of Staphylococcus aureus, on various Proteus strains and other Gram-negative bacteria, including carbenicillin-sensitive and R + strains of Pseudomonas aeruginosa and potent P-Iactamase producers in Escherichia coli R+TEM, Enterobacter cloacae P99 and Klebsiella aerogenes K l (Marshall, Ross, Ghanter & Harris, 1972). In addition, the effects of cefamandole on growth, lysis and induction of morphological variants in some strains have been studied. Materials and methods Bacterial strains These consisted of penicillin-susceptible and ^-lactamase producing strains of S. aureus and various strains of Ps. aeruginosa and other Gram-negative bacteria. All strains were grown overnight in nutrient broth (Oxoid) at 37°C. Minimum inhibitory concentrations (MICs) MIC values were determined by placing 0-05 ml of a 1 in 100 dilution (c. 5 X 10' viable cells/ml) of an overnight culture of an organism on to the surface of overdried agar 97
Downloaded from http://jac.oxfordjournals.org/ at Indiana University Library on July 10, 2015
Welsh School of Pharmacy, University of Wales Institute of Science and Technology, Cathays Park, Cardiff, Wales
98
A. D. Russell
plates containing the desired cefamandole concentration. The plates were incubated at 37°C for 24 h and examined for the presence or absence of growth. The minimum concentration completely preventing growth was taken as the MIC.
N
COOH
N
N N I CH 3
Figure 1. Chemical structure of cefamandole.
Results and discussion The MIC values of cefamandole against various strains of S. aureus and of Gramnegative bacteria are shown in Tables I and II. Although only a limited number of strains has been tested the following conclusions may be tentatively reached: (a) cefamandole is active against penicillin-sensitive and ^-lactamase producing strains of S. aureus. Inoculum size appears to have only a slight effect, if any; (b) non beta-lactamase producing strains of E, coli (NCTC 9001, NCTC 100418 and R~TEM) are sensitive to cefamandole, whereas the only p-lactamase producer tested (E. coli R+TEM) is highly resistant; (c) other Gram-negative bacteria which are powerful producers of j5-lactamase (Enterobact. cloacae P99 and K. aerogenes Kl) are also highly resistant to cefamandole (cf. the non-fi-lactamase-producing mutant, Enterobact. cloacae P99M, in Table II); (d) all strains of Ps. aeruginosa tested, irrespective of whether or not they are carbenicillin sensitive or possess R-factors, are highly resistant to cefamandole; (e) all strains of Pr. mirabilis and some strains of Pr. rettgeri and Pr. morganii are sensitive to cefamandole, whereas the strains of Pr. vulgaris are resistant. Cephaloridine shows the same pattern of response with the strains listed in (b) and (c) above (Russell, 1972), but is particularly prone to changes in inoculum size with staphylococci under (a) (Ridley & Phillips, 1965; Benner, Bennett, Broadie & Kirby, 1965). Cephaloridine is also active against most strains of Pr. mirabilis (Eykyn, 1971), and thus the two antibiotics show a similar response in this context The effects of adding cefamandole to growing cultures of various strains are shown in Figures 2 to 7. The antibiotic induces lysis of P99M (Figure 2) and R~TEM (Figure 3)
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Effect of cefamandole on rate of growth 0-1 ml vol. of overnight 37°C broth cultures were added to 9-4 ml of nutrient broth in nephelos flasks (Pyrex flasks with attached side-arms) previously equilibrated at 37°C in a shaking incubator (100 oscillations/min). When the cultures had entered the logarithmic phase of growth at 37°C, 0-5 ml of differing cefamandole solutions were added to give the desired final drug concentration. Incubation was continued at 37°C. Turbidity changes before and after the addition of the antibiotic were monitored with the EEL Unigalvo. In cases where the turbidity of cultures continued to increase after the addition of cefamandole, samples were removed for microscopical examination (phase contrast microscope, x 900). Photographs of these samples were taken by means of a Kodak camera attached to a Vickers microscope.
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Plate 1. Morphological changes in some Gram-negative bacteria exposed to cefamandole at stated concentration, (a) Pr. mirabilis 10374, 20 ng/ml, 250min; (b)Pr. mirabilis 10374, 10 ng/ml, 250 min; (c)Pr. vulgaris4M5, 20tig/ml, 250min; (d) Pr. rettgeri 8893, 20 ng/ml, 270min; (e)Pr. rettgeri8893, 10(ig/ml, 270 min; (0 Pr. morganii 235, 20 tig/ml, 250 min; (g) E. co// R"TEM, lOjig/ml, 300 min; (h) Enterobact. cloacae P99M, 20 ng/ml, 300 min; (i) P99M, 10 ng/ml, 300 min.
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Antimicrobial activity of a new cephalosporin, cefamandole
99
Table I. MIC values for cefamandole against some strains of staphylococci MIC of cefamandole (ng/ml) Organism S. aureus
Oxford NCTC 3761 1605* 3452* 11127* 1613*
A
B
C
0-5 0-5 5 0-5 0-5 10
5 0-5 0-5 2-5
2-5 0-5 0-5 2-5
but a concentration of 20 ng/ml, or about 4 to 8 times the respective MIC, is needed before this is achieved. Concentrations of up to 20 |xg/ml were also tested with other strains; as would be expected from the MIC values in Table n , 20 ji-g/ml cefamandole had a negligible effect on the growth of R + T E M and P99. However, cefamandole at 20 p-g/ml did not cause lysis or markedly reduce the growth rate of Pr. mirabilis NCTC 10374 (Figure 4) or of the highly ampicillin-sensitive Pr. morganii NCTC 235 (Figure 5); some lysis of Pr. rettgeri NCTC 8893 occurred at 5 and 10 (xg/ml, followed by re-growth, with a rather more extensive lysis at 20 ng/ml (Figure 6). Surprisingly, cefamandole at 10 and especially 20 ng/ml, reduced the rate of growth of Pr. vulgaris NCTC 4175, but no lysis occurred (Figure 7) and, at 24-h incubation, extensive growth of this organism had occurred in all concentrations. Samples were removed from those flasks which continued to show an increase in Table n . MIC values for cefamandole against some strains of Gram-negative bacteria Strain
Organism Pr. vulgaris
Pr. morganii Pr. rettgeri
NCTC 10015 NCTC 4175 NCTC 10376 J. 27* NCTC 235 NCTC 1707 NCTC 5845 NCTC 8893 NCTC 7475 NCTC 10371 J23* H* H2*
Pr. mirabilis
NCTC 6369 NCTC 10374 NCTC 9589 S4*
S39* S33*
MIC ((ig/ml) 100 100
>100 100 7-5 5 100 2-5 10 100 7-5 15 50 7-5
Organism
Strain
Ps. aeruginosa R+3425 R+1822 NCTC 6750 NCTC 8203 NCTC 10701 NCTC 10490 NCTC 1999 E. coli NCTC 9001 NCTC 100418 R-TEM R+TEM Enterobact cloacae
P99 P99M
MIC (ng/ml) >1000 >1000 >1000 >1000 >1000 >1000 >1000 2-5 1 1 500 500 5
2-5-5 5 25 2-5 2-5
Kl. aerogenes Kl
* Hospital isolates. CEM cefamandole.
>500
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* ()-lactamase producers. A, B, C, . . . 1 in 100, 1 in 10,000 and 1 in 1,000,000 dilutions,respectivelyof an overnight culture.
A. D. Russell 100
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Figures 2-7
2
0 Tlmeot37#C(h)
6
Antimicrobial activity of a new cephalosporin, cefamandole
101
Acknowledgements The author thanks Dr C. F. Speirs of Lilly Research Ltd, Windlesham for generous supplies of cefamandole; Kodak Ltd, for financial help with the photomicrographic studies; and Mrs J. A. Harris, Miss L. M. Smart and Mr D. Collins for technical assistance. References Benner, E. J., Bennett, J. V., Broadie, J. L. & Kirby, W. M. M. Inactivation of cephalothin and cephaloridine by Staphylococcus aureus. Journal of Bacteriology 90: 1599-1604 (1965). Eykyn, S. Use and control of cephalosporins. Journal of Clinical Pathology 24: 419-29 (1971). Eykyn, S., Jenkins, C , King, A. & Phillips, I. Antibacterial activity of cefamandole, a new cephalosporin antibiotic, compared with that of cephaloridine, cephalothin and cephalexin. Antimicrobial Agents and Chemotherapy 3: 657-61. Marshall, M. J., Ross, G. W., Ghanter, K. V. & Harris, A. M. Comparison of the substrate specifities of the P-lactamase from Klebsiella aerogenes 1082E and Enterobacter cloacae P99. Applied Microbiology 23: 765-9 (1972). Ridley, M. & Phillips, I. Relative instability of cephaloridine to Staphylococcal penicillinase. Nature, London 208: 1076-8 (1965). Russell, A. D. In vitro studies with CIBA 36,278-Ba, a new cephalosporin derivative. Microbios 6: 221-30. (1972). Russell, A. D. Effect of some p-lactam antibiotics on the morphology of some P-lactamase and non-P-lactamase producing strains of Gram-negative bacteria. Journal of Applied Bacteriology 36: 357-9 (1973). Wick, W. E. & Preston, D. A. Biological properties of three 3-heterocyclic-thiomethylcephalosporin antibiotics. Antimicrobial Agents for Chemotherapy 1: 221-34 (1972). [Manuscript accepted 19 November 1974)
Figure 2. Effect of cefamandole (added at 4 ) on the growth of E. coli R"TEM in broth at 37°C. Cefamandole concentrations (ng/ml): 0, A—A; 2-5, • — • ; 5, o—O; 10, • — • ; 20, D—D. Figure 3. Effect of cefamandole (added at | ) on the growth of Enterobact. cloacae P99M in broth at 37°C. Symbols: as in Figure 2. Figure 4. Effect of cefamandole (added at j ) on the growth of Pr. mirabilis N.C.T.C. 10374 in broth at 37°C. Symbols as in Figure 2. Figure 5. Effect of cefamandole (added at j ) on the growth of Pr. morganil N.C.T.C. 235 in broth at 37°C. Symbols as in Figure 2. Figure 6. Effect of cefamandole (added at | ) on the growth of Pr. rettgeri N.C.T.C. 8893 in broth at 37°C. Symbols as in Figure 2. Figure 7. Effect of cefamandole (added at | ) on the growth of Pr. vulgaris N.C.T.C. 4175 in broth at 37°C. S>mbols as in Figure 2.
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turbidity and examined by phase-contrast microscopy. Examples of the morphological forms induced by cefamandole are shown in Plate 1. These indicate that increases in turbidity may be the result of morphological variants, which are typical of those induced by p-lactam antibiotics in p-lactamase and non-p-lactamase producing Gram-negative bacteria (Russell, 1973). It must be pointed out that much higher numbers of viable bacteria are of necessity present in these growth experiments than in the MIC experiments described in Tables I and II. Thus, bearing this in mind, it may be concluded (i) that inoculum size may be of importance with these Gram-negative strains, (ii) that the minimum lytic concentration, which would correspond to a minimum bactericidal concentration (MBC) with a [J-lactam antibiotic, is several times higher than the MIC. This latter point confirms the recent finding made by Eykyn, Jenkins, King & Phillips (1973) for cefamandole and a wider range of Gram-negative bacteria.
