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Affinity of Cefmenoxime for Beta-Lactamases: An Analysis
Affinity of Cefmenoxime for Beta-Lactamases: An Analysis
The interactions of cefmenoxime with beta-lactamases in comparison with cefotaxime, moxalactam, cefoperazone, and ceftazidime have been determined. On-line computerized microacidimetry allowed determination of the affinity of these compounds with the enzymes, which was characterized by Km values. The beta-lactamases that were used were two cephalosporinases and one penicillinase. Within these data, the cephalosporins could be classified into three groups: (1) those with high affinity for the cephalosporinases and very poor affinity for the penicillinase (cefmenoxime, cefotaxime, and moxalactam); (2) those with moderate affinity for the cephalosporinases and very poor affinity for the penicillinase (ceftazidime); (3) those with poor affinity for all enzymes (cefoperazone). In the case of the penicillinase {TEI\II-1), only cefoperazone was subject to some hydrolysis.
ROGER LABIA, Ph.D. ANNICK MORAND CATHERINE VERCHERE-BEAUR, Ph.D. Paris, France
ANDRE BRYSKIER, M.D. Argenteuil, France
Cefmenoxime, a new third-generation cephalosporin, has a broad antibacterial spectrum, dealing mostly with gram-negative bacilli [1-3]. In our studies on the mode of action of beta-lactam antibiotics, we determined the interaction of cefmenoxime with a few beta-lactamases produced by gram-negative bacilli. These are representative of the various enzymes produced by the microorganisms usually or frequently involved in human infectious diseases. Comparisons were made with cefotaxime, moxalactam, cefoperazone, and ceftazidime.
MATERIALS AND METHODS
From the Museum National d'Histoire NaturelleC.N.R.S. (UA 401), Paris, France, and Laboratoire de Bacteriologie, C.H.G., Argenteuil, France. Requests for reprints should be addressed to Dr. R. Labia, Museum National d'Histoire Naturelle, 63, rue Button, 75005 Paris, France.
Cefmenoxime was compared with cefotaxime, moxalactam, cefoperazone, and ceftazidime and, in some instances, with cephalothin. In the assessment of kinetic constants, penicillin G was used as a reference and for comparative purposes, was given an arbitrary value of 100 for maximum rate of hydrolysis. The plasmid mediated beta-lactamase (penicillinase) TEM-1 (pi= 5.4) was obtained from cultures of Escherichia coli K-12 carrying the plasmid P 111 [4]. The chromosomally mediated beta-lactamases (cephalosporinases) were obtained from Morganella morganii GN 1510 (pi= 7.3) [5] and Proteus vulgaris RO 104 (pi= 8.7) (Dr. Pitton, Geneva, Switzerland). The interactions of the beta-lactam antibiotics with beta-lactamases had been monitored by on-line computerized microacidimetry. Within "good substrates," that is, those rapidly hydrolyzed, this method allows simultaneous determination of the two Michaelis-Menten constants: Km (affinity of the substrate for the enzyme) and Vm (maximum rate of hydrolysis). With products that are poorly hydrolyzed, their affinity for the enzymes can also be determined, but only by competition experiments. In the present study, the Vm values are given in relation to penicillin G hydrolysis, that is, Vm = 100 [4,6]. Sometimes these values are given in relation to cephalothin; the corresponding data for this antibiotic will allow these determinations if desired.
December 21, 1984
The American Journal of Medicine
Volume
n
(suppl 6A)
25
SYMPOSIUM ON CEFMENOXIME-LABIA ET AL
TABLE I
Kinetic Constants of the Various BetaLactams towards Beta-Lactamases Morganella morganii (Cef)
TEM-1 (Pen) Drug Cefmenoxime Cefotaxime Moxalactam Ceftazidime Cefoperazone Cephalothin Penicillin G
Km*
vmt
Km
40 10 100
0.05 0.04 0.02 8.8 4.0 95 3.5
i
270 230 21
Vm
Proteus vulgaris (Caf)
Km 185 185 40
2.5 400 100
9.0 26 3.1
Vm 390 390
150 3,600 100
Km = affinity of the substrate for the enzyme; Pen = penicillinase; Vm = maximum rate of hydrolysis; Cef = cephalosporinase. *Km is given in micromoles. tvm is related to penicillin G (100). *Not measurable: poor affinity or poor hydrolysis.
RESULTS AND COMMENTS
Our experiments involved a large set of beta-lactamases, but for clarity we list here the results obtained with only a few of them. Nevertheless, the results have general significance. The enzymes reported on are (1) the TEM-1 Afactor mediated penicillinase; (2) a chromosomally mediated cephalosporinase produced by M. morganii; (3) a less common cephalosporinase produced by P; vulgaris, which shows an increased hydrolytic activity into methoxy-imino cephalosporins, such as cefotaxime. This broad spectrum beta-lactamase is also chromosomally mediated. Cefmenoxime, cefotaxime, moxalactam, and ceftazidime show practically no interaction with the TEM-1 penicillinase. This situation corresponds to very poor affinity, if any: very low hydrolysis, if any (Table 1). Cefoperazone is subject to some hydrolysis by the TEM-1 penicillinase; it shows a Vm of 40 (related to penicillin G), but its affinity for the enzyme is moderate (Km = 270 p.M). The affinity is, for example, about 10 times lower than that of a good substrate such as ampicillin (Km = 29 p.M). It ·is known that on TEM-1 or TEM-Iike producing strains, cefoperazone antibacterial activity is somewhat reduced in comparison with that of nonpenicillinase-producing strains [7]. Cephalosporinases are a class of beta-lactamases usually produced by a large number of Enterobacteriaceae
such as indole-positive Proteus, Enterobacter, Serratia, and Citrobacter. These strains are usually resistant to the first generation cephalosporins, such as cephalothin, which are good substrates for the enzymes (that is, subject to rapid degradation). The M. morganii cephalosporinase appeared representative of the group. Cefmenoxime, cefotaxime, and moxalactam demonstrate a very high affinity for the M. morganii beta-lactamase, as shown by very low Km values. Nevertheless, their hydrolysis is still poor; very low Vms are difficult to measure with accuracy. Previously [8], we showed that moxalactam had the ability to inactivate cephalosporinases, an unusual property not evidenced by the other presently tested compounds. Surprisingly, ceftazidime has a much lower affinity for this enzyme, as its Km value is more than 100 times higher than that of cefmenoxime, cefotaxime, or moxalactam. As demonstrated in the previous cephalosporins, ceftazidime hydrolysis also is not detectable by the acidimetric technique. Cefoperazone has a moderate affinity for the M. morganii cephalosporinase and in this aspect is similar to ceftazidime; nevertheless, a low rate of hydrolysis can be detected. The last cephalosporinase, produced by P. vulgaris, is similar in many ways to the previous cephalosporinase. Nevertheless, interesting differences are evident, mainly a high sensitivity to the inhibitory action of clavulanic acid and an important hydrolysis of the methoxy-imino cephalosporins such as cefuroxime and cefotaxime. This enzyme usually has an inducible and low level of biosynthesis, which could explain why cefotaxime and cefmenoxime are often very active against this bacterial species. With this P. vulgaris cephalosporinase, cefmenoxime demonstrates the same behavior as cefotaxime, that is, practically the same affinity and the same rate of hydrolysis. Moxalactam has a slightly stronger affinity, but, as with the previous enzyme, it is still an inactivator [8]. Ceftazidime has poor interactions with the enzyme, whereas cefoperazone has a much better affinity than cefmenoxime or cefotaxime and a lower rate of hydrolysis. In conclusion, among the enzymes cited herein, cefmenoxime showed a behavior very close to that of cefotaxime: (1) practically no interactions with penicillinases; (2) good affinity but poor hydrolysis for the "usual" cephalosporinases; (3) some hydrolysis by the less common "extended-spectrum" cephalosporinases, such as that produced by P. vulgaris, but poor affinity.
REFERENCES 1.
26
Tsuchiya K, Kita Y, Yamazaki T, Kondo M, Noji Y, Fugono T: Absorption, distribution and excretion of cefmenoxime (SCE 1365), a new broad-spectrum cephalosporin, in mice, rats, rabbits and dogs. J Antibiotic 1980; 33: 1532-1544.
December 21, 1984
The American Journal of Medicine
2.
Tsuchiya K, Kondo M, Kida M, et al: Cefmenoxime (SCE 1365), a novel broad-spectrum cephalosporin: in vitro and in vivo antibacterial activities. Antimicrob Agents Chemother 1981; 19: 56-65.
Volume 77 (suppl 6A)
SYMPOSIUM ON CEFMENOXIME-LABIA ET AL
3. 4.
5.
Stamm JM, Girolami RL, Shipkowitz NL, Bower RR: Antibacterial activity of cefmenoxime (SCE 1365). Antimicrob A!:Jents Cl1emother 1981; 19: 454-460. Labia R, Barthelemy M. Fabre C, Guionie M, Peduzzi J: Comparative studies of three A-factor mediated beta-lactamases. In: Hamilton-Miller JMT, Smith JT, eds. Beta-lactamases. London: Academic Press, 1979; 429-442. Fujii-Kuriyama Y, Yamamoto M, Sugawara S: Purification and properties of beta-lactamase from Proteus morganii. J Bacte-
December 21, 1984
6. 7. 8.
riol 1977; 131: 726-734. Labia R: Comportement enzyme-substrat. Introduction de Ia notion de stabilite enzymatique dans le cas des beta-lactamases. CR Acad Sci 1974; 2790: 109-112. Chabbert YA, Derlot E: Comparative activity of cefotetan on Escherichia coli K-12 possessing plasmid mediated beta-lactamases. J Antimicrob Chemother 1983; 11 (suppl A): 159-167. Labia R: Moxalactam: an oxa-beta-lactam antibiotic that inactivates beta-lactamases. Rev Infect Dis 1982; 4 (suppl): 529.
The American Journal of Medicine
Volume
n
(suppl 6A)
27
The interactions of cefmenoxime with beta-lactamases in comparison with cefotaxime, moxalactam, cefoperazone, and ceftazidime have been determined. On-line computerized microacidimetry allowed determination of the affinity of these compounds with the enzymes, which was characterized by Km values. The beta-lactamases that were used were two cephalosporinases and one penicillinase. Within these data, the cephalosporins could be classified into three groups: (1) those with high affinity for the cephalosporinases and very poor affinity for the penicillinase (cefmenoxime, cefotaxime, and moxalactam); (2) those with moderate affinity for the cephalosporinases and very poor affinity for the penicillinase (ceftazidime); (3) those with poor affinity for all enzymes (cefoperazone). In the case of the penicillinase {TEI\II-1), only cefoperazone was subject to some hydrolysis.
ROGER LABIA, Ph.D. ANNICK MORAND CATHERINE VERCHERE-BEAUR, Ph.D. Paris, France
ANDRE BRYSKIER, M.D. Argenteuil, France
Cefmenoxime, a new third-generation cephalosporin, has a broad antibacterial spectrum, dealing mostly with gram-negative bacilli [1-3]. In our studies on the mode of action of beta-lactam antibiotics, we determined the interaction of cefmenoxime with a few beta-lactamases produced by gram-negative bacilli. These are representative of the various enzymes produced by the microorganisms usually or frequently involved in human infectious diseases. Comparisons were made with cefotaxime, moxalactam, cefoperazone, and ceftazidime.
MATERIALS AND METHODS
From the Museum National d'Histoire NaturelleC.N.R.S. (UA 401), Paris, France, and Laboratoire de Bacteriologie, C.H.G., Argenteuil, France. Requests for reprints should be addressed to Dr. R. Labia, Museum National d'Histoire Naturelle, 63, rue Button, 75005 Paris, France.
Cefmenoxime was compared with cefotaxime, moxalactam, cefoperazone, and ceftazidime and, in some instances, with cephalothin. In the assessment of kinetic constants, penicillin G was used as a reference and for comparative purposes, was given an arbitrary value of 100 for maximum rate of hydrolysis. The plasmid mediated beta-lactamase (penicillinase) TEM-1 (pi= 5.4) was obtained from cultures of Escherichia coli K-12 carrying the plasmid P 111 [4]. The chromosomally mediated beta-lactamases (cephalosporinases) were obtained from Morganella morganii GN 1510 (pi= 7.3) [5] and Proteus vulgaris RO 104 (pi= 8.7) (Dr. Pitton, Geneva, Switzerland). The interactions of the beta-lactam antibiotics with beta-lactamases had been monitored by on-line computerized microacidimetry. Within "good substrates," that is, those rapidly hydrolyzed, this method allows simultaneous determination of the two Michaelis-Menten constants: Km (affinity of the substrate for the enzyme) and Vm (maximum rate of hydrolysis). With products that are poorly hydrolyzed, their affinity for the enzymes can also be determined, but only by competition experiments. In the present study, the Vm values are given in relation to penicillin G hydrolysis, that is, Vm = 100 [4,6]. Sometimes these values are given in relation to cephalothin; the corresponding data for this antibiotic will allow these determinations if desired.
December 21, 1984
The American Journal of Medicine
Volume
n
(suppl 6A)
25
SYMPOSIUM ON CEFMENOXIME-LABIA ET AL
TABLE I
Kinetic Constants of the Various BetaLactams towards Beta-Lactamases Morganella morganii (Cef)
TEM-1 (Pen) Drug Cefmenoxime Cefotaxime Moxalactam Ceftazidime Cefoperazone Cephalothin Penicillin G
Km*
vmt
Km
40 10 100
0.05 0.04 0.02 8.8 4.0 95 3.5
i
270 230 21
Vm
Proteus vulgaris (Caf)
Km 185 185 40
2.5 400 100
9.0 26 3.1
Vm 390 390
150 3,600 100
Km = affinity of the substrate for the enzyme; Pen = penicillinase; Vm = maximum rate of hydrolysis; Cef = cephalosporinase. *Km is given in micromoles. tvm is related to penicillin G (100). *Not measurable: poor affinity or poor hydrolysis.
RESULTS AND COMMENTS
Our experiments involved a large set of beta-lactamases, but for clarity we list here the results obtained with only a few of them. Nevertheless, the results have general significance. The enzymes reported on are (1) the TEM-1 Afactor mediated penicillinase; (2) a chromosomally mediated cephalosporinase produced by M. morganii; (3) a less common cephalosporinase produced by P; vulgaris, which shows an increased hydrolytic activity into methoxy-imino cephalosporins, such as cefotaxime. This broad spectrum beta-lactamase is also chromosomally mediated. Cefmenoxime, cefotaxime, moxalactam, and ceftazidime show practically no interaction with the TEM-1 penicillinase. This situation corresponds to very poor affinity, if any: very low hydrolysis, if any (Table 1). Cefoperazone is subject to some hydrolysis by the TEM-1 penicillinase; it shows a Vm of 40 (related to penicillin G), but its affinity for the enzyme is moderate (Km = 270 p.M). The affinity is, for example, about 10 times lower than that of a good substrate such as ampicillin (Km = 29 p.M). It ·is known that on TEM-1 or TEM-Iike producing strains, cefoperazone antibacterial activity is somewhat reduced in comparison with that of nonpenicillinase-producing strains [7]. Cephalosporinases are a class of beta-lactamases usually produced by a large number of Enterobacteriaceae
such as indole-positive Proteus, Enterobacter, Serratia, and Citrobacter. These strains are usually resistant to the first generation cephalosporins, such as cephalothin, which are good substrates for the enzymes (that is, subject to rapid degradation). The M. morganii cephalosporinase appeared representative of the group. Cefmenoxime, cefotaxime, and moxalactam demonstrate a very high affinity for the M. morganii beta-lactamase, as shown by very low Km values. Nevertheless, their hydrolysis is still poor; very low Vms are difficult to measure with accuracy. Previously [8], we showed that moxalactam had the ability to inactivate cephalosporinases, an unusual property not evidenced by the other presently tested compounds. Surprisingly, ceftazidime has a much lower affinity for this enzyme, as its Km value is more than 100 times higher than that of cefmenoxime, cefotaxime, or moxalactam. As demonstrated in the previous cephalosporins, ceftazidime hydrolysis also is not detectable by the acidimetric technique. Cefoperazone has a moderate affinity for the M. morganii cephalosporinase and in this aspect is similar to ceftazidime; nevertheless, a low rate of hydrolysis can be detected. The last cephalosporinase, produced by P. vulgaris, is similar in many ways to the previous cephalosporinase. Nevertheless, interesting differences are evident, mainly a high sensitivity to the inhibitory action of clavulanic acid and an important hydrolysis of the methoxy-imino cephalosporins such as cefuroxime and cefotaxime. This enzyme usually has an inducible and low level of biosynthesis, which could explain why cefotaxime and cefmenoxime are often very active against this bacterial species. With this P. vulgaris cephalosporinase, cefmenoxime demonstrates the same behavior as cefotaxime, that is, practically the same affinity and the same rate of hydrolysis. Moxalactam has a slightly stronger affinity, but, as with the previous enzyme, it is still an inactivator [8]. Ceftazidime has poor interactions with the enzyme, whereas cefoperazone has a much better affinity than cefmenoxime or cefotaxime and a lower rate of hydrolysis. In conclusion, among the enzymes cited herein, cefmenoxime showed a behavior very close to that of cefotaxime: (1) practically no interactions with penicillinases; (2) good affinity but poor hydrolysis for the "usual" cephalosporinases; (3) some hydrolysis by the less common "extended-spectrum" cephalosporinases, such as that produced by P. vulgaris, but poor affinity.
REFERENCES 1.
26
Tsuchiya K, Kita Y, Yamazaki T, Kondo M, Noji Y, Fugono T: Absorption, distribution and excretion of cefmenoxime (SCE 1365), a new broad-spectrum cephalosporin, in mice, rats, rabbits and dogs. J Antibiotic 1980; 33: 1532-1544.
December 21, 1984
The American Journal of Medicine
2.
Tsuchiya K, Kondo M, Kida M, et al: Cefmenoxime (SCE 1365), a novel broad-spectrum cephalosporin: in vitro and in vivo antibacterial activities. Antimicrob Agents Chemother 1981; 19: 56-65.
Volume 77 (suppl 6A)
SYMPOSIUM ON CEFMENOXIME-LABIA ET AL
3. 4.
5.
Stamm JM, Girolami RL, Shipkowitz NL, Bower RR: Antibacterial activity of cefmenoxime (SCE 1365). Antimicrob A!:Jents Cl1emother 1981; 19: 454-460. Labia R, Barthelemy M. Fabre C, Guionie M, Peduzzi J: Comparative studies of three A-factor mediated beta-lactamases. In: Hamilton-Miller JMT, Smith JT, eds. Beta-lactamases. London: Academic Press, 1979; 429-442. Fujii-Kuriyama Y, Yamamoto M, Sugawara S: Purification and properties of beta-lactamase from Proteus morganii. J Bacte-
December 21, 1984
6. 7. 8.
riol 1977; 131: 726-734. Labia R: Comportement enzyme-substrat. Introduction de Ia notion de stabilite enzymatique dans le cas des beta-lactamases. CR Acad Sci 1974; 2790: 109-112. Chabbert YA, Derlot E: Comparative activity of cefotetan on Escherichia coli K-12 possessing plasmid mediated beta-lactamases. J Antimicrob Chemother 1983; 11 (suppl A): 159-167. Labia R: Moxalactam: an oxa-beta-lactam antibiotic that inactivates beta-lactamases. Rev Infect Dis 1982; 4 (suppl): 529.
The American Journal of Medicine
Volume
n
(suppl 6A)
27