Vol. 34, No. 4
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1990, p. 555-561
0066-48041901040555-07$02.00/0 Copyright © 1990, American Society for Microbiology
Antimicrobial Activity and Stability to 1-Lactamase of BMY-28271, a New Oral Cephalosporin Ester HIROYUKI MATSUI,'* MASAKI HIRAOKA,2 MATSUHISA INOUE,3 AND SUSUMU MITSUHASHI' Episome Institute, 2220 Kogure, Fujimi-mura, Seta-gun, Gunma 371-01,1 Preclinical Research Laboratories, Bristol-Myers Research Institute, Ltd., I Futagoyama, Sakazaki, Kota-cho, Nukata-gun, Aichi 444_0J,2 and Laboratory of Drug Resistance in Bacteria, School of Medicine, Gunma University, 39-22, Showa-machi, Maebashi, Gunma 37J,3 Japan Received 17 July 1989/Accepted 21 December 1989
BMY-28271, the acetoxyethyl ester of BMY-28232, 7-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-3(Z)-propen-l-yl-3-cephem-4-carboxylic acid, is a new oral cephalosporin. BMY-28232 has a widely expanded spectrum with high activity against gram-positive and gram-negative bacteria. BMY-28232 is far more active than cefixime or cefteram against Staphylococcus aureus and Staphylococcus epidermidis. Against gram-negative bacteria, the activity of BMY-28232 was comparable to or somewhat weaker than that of cefixime or cefteram. BMY-28232 was a poor substrate for various P-lactamases. Orally administered BMY-28271 had a good therapeutic effect on systemic infections with S. aureus and some gram-negative bacteria in mice. Oral BMY-28271 was efficacious against S. aureus Smith infection: the efficacy of BMY-28271 was 80 to 90 times higher than that of cefixime or cefteram.
Many cephalosporins with broad antibacterial spectra have been used clinically for the treatment of a wide range of bacterial infections. Recently, there has been great progress in the development of oral cephalosporins, but each clinically available cephalosporin has weaknesses. Cefaclor is active against gram-positive and some gram-negative bacteria but is hydrolyzed by various f-lactamases (13). Cefixime and cefteram (T-2525) (2, 12, 14) have good activity against gram-negative bacteria and streptococci and are not hydrolyzed readily by various P-lactamases, but these compounds have relatively low activities against staphylococci (2, 6, 12, 14). BMY-28232, the parent compound of BMY-28271, is active against gram-positive and gram-negative bacteria and is relatively resistant to hydrolysis by various 1-lactamases (16). The present study deals with the in vitro antibacterial activity and P-lactamase stability of BMY-28232 and the in vivo antibacterial activity of BMY-28271 in comparison with those of cefixime and cefteram.
Determination of MICs. MICs were determined by the twofold agar dilution method. The media used for preculture and MIC determination were as follows: brain heart infusion broth (Difco Laboratories, Detroit, Mich.) and sensitivity disk agar (SDA; Nissui Seiyaku Co., Ltd., Tokyo, Japan) supplemented with 5% defibrinated horse blood for streptococci; brain heart infusion broth and SDA supplemented with 5% Fildes enrichment (Difco) for Haemophilus influenzae; sensitivity test broth (STB; Nissui Seiyaku) supplemented with 0.4% KNO3 and SDA for Pseudomonas spp.; GAM broth and agar (Nissui) for anaerobes; and STB and SDA for the other microorganisms. Organisms were precultured for 18 h at 37°C and suitably diluted in buffered saline (pH 7.0) containing 0.01% gelatin. Approximately 104 CFU of bacteria per spot were inoculated onto drug-containing agar plates with a multiloop inoculator (Microplanter; Sakuma Seisakusho Co., Ltd., Tokyo, Japan). Plates were incubated for 18 h at 37°C, except for obligate anaerobes, which were incubated for 48 h. Haemophilus influenzae and Neisseria gonorrhoeae were incubated in a candle jar, and anaerobes were incubated in an anaerobic jar. The MIC was defined as the lowest drug concentration which prevented visible growth of bacteria. Hydrolysis by 13-lactamases. The various types of P-lactamases (1, 11) used in this study were purified and stored at -80°C at the Episome Institute (3-5, 9, 18, 19). Hydrolysis of the drugs was assayed by a spectrophotometric method (17) at a substrate concentration of 100 ,uM. The susceptibility of the drugs to hydrolysis by various P-lactamases was expressed as the rate of hydrolysis relative to that of cephaloridine or benzylpenicillin, which was set at 100. For the 3-lactamases of Enterobacter cloacae GN7471 (10), Citrobacter freundii GN7391 (15), and Proteus vulgaris GN7919 (7), Km and Vmax values were determined from hydrolytic rates at various substrate concentrations by use of the Lineweaver-Burke plot. Bactericidal activity. An overnight shaking culture of organisms in STB at 37°C was inoculated into freshly prepared STB, and the culture was shaken at 37°C for 2 h. When the organisms had grown to a density of approximately 105
MATERIALS AND METHODS Antibiotics. BMY-28232 and BMY-28271 (the acetoxyethyl ester of BMY-28232) were synthesized at the Tokyo Research Center of Bristol Myers Research Institute, Ltd., Tokyo, Japan.- The other antimicrobial agents used in this study were obtained from the indicated sources: cefixime, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan; cefteram (T-2525, Ro 19-5247) and cefteram pivoxil (T-2588) (2, 12, 14), Toyama Chemical Co., Ltd., Tokyo, Japan; cefaclor, Shionogi Pharmaceutical Co., Ltd., Osaka, Japan; benzylpenicillin, Meiji Seika Kaisha, Ltd., Tokyo, Japan; cephaloridine, Nihon Glaxo Co., Ltd., Tokyo, Japan; and methicillin, Banyu Pharmaceutical Co., Ltd., Tokyo, Japan. Organisms. The bacterial strains used in this study were recent clinical isolates from various hospitals in Japan. The 3-lactamase-producing strains used in this study were maintained at the Episome Institute. All strains were stored at -800C. *
Corresponding author. 555
556
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ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. In vitro antibacterial activity of BMY-28232 against clinical isolates Organism (no. of strains)
Antibiotic
MIC Range
(4m1)a 50%
90%
Methicillin-susceptible Staphylococcus aureus (54)
BMY-28232 Cefixime Cefteram
0.39-0.78 6.25-25 3.13-12.5
0.78 12.5 6.25
0.78 25 6.25
Methicillin-susceptible Staphylococcus epidermidis (30)
BMY-28232 Cefixime Cefteram
0.025-3.13 3.13-100 0.39-12.5
0.10 6.25 1.56
0.39 50 6.25
Methicillin-resistant Staphylococcus epidermidis (39)
BMY-28232 Cefixime Cefteram
0.05->100 12.5->100 3.13->100
Streptococcus pyogenes (99)
BMY-28232 Cefixime Cefteram
100
25 100 50
1.56 0.78 0.78 >100 >100 >100 12.5 3.13 25
25 100 >100
>100 >100 >100
a 50o and 90%o, MICs for 50 and 90%o of strains tested, respectively. b Inoculum of 106 CFU per spot.
CFU/ml, antibiotics were added to the culture at concentrations ranging from one-fourth to four times the MIC determined by the agar dilution method. Samples were taken at intervals and inoculated onto drug-free heart infusion agar plates; the numbers of colonies were counted after 18 h of incubation at 37°C. Determination of oral in vivo activity. The in vivo antibacterial activities of the drugs were determined against systemic infections in mice. Ten 4-week-old male STD:ddY mice (weight, 19 to 21 g) were used for each dose of drug. An overnight culture on heart infusion agar at 37°C was suspended in physiological saline for Escherichia coli ML4707 and Klebsiella pneumoniae GN6445 or in 5% gastric mucin for Staphylococcus aureus Smith and Proteus mirabilis GN4754. A 0.5-ml portion of the bacterial suspension was inoculated intraperitoneally into each mouse. Under these conditions, all untreated mice died within 5 days. The drugs were suspended in 0.5% carboxymethyl cellulose and were given to the mice as a single oral dose immediately after infection. The 50% effective dose was calculated by the Probit method (8) from the number of mice that survived at each dose level 5 days after bacterial challenge.
RESULTS Antimicrobial activity against clinical isolates. The in vitro antimicrobial activity of BMY-28232 against gram-positive and gram-negative organisms was compared with those of cefixime and cefteram (T-2525, Ro 19-5247). Table 1 shows the concentrations of antibiotics required to inhibit the growth of 50 and 90%o (MIC%0) of the total number of strains tested. The MIC90s of BMY-28232 against gram-positive organisms such as methicillin-susceptible Staphylococcus aureus, methicillin-susceptible Staphylococcus epidermidis, Streptococcus pyogenes, and Streptococcus pneumoniae ranged from 0.025 to 0.78 ,ug/ml. BMY-28232 was 32- to 128-fold more active than cefixime and 8- to 16-fold more active than cefteram against these staphylococci. Although BMY-28232 was active against some strains of methicillinresistant Staphylococcus epidermidis, most of the methicillin-resistant staphylococci were resistant to BMY-28232 and the other two agents. BMY-28232 was active (MIC90s, 0.025 and 0.10 ,ug/ml, respectively) against Streptococcus pyogenes and Streptococcus pneumoniae. The MICs of BMY28232 against Enterococcus faecalis were distributed in a
558
MATSUI ET AL.
ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Activity of BMY-28232 against ,-lactamase producing bacteria MIC
,3-Lactamase-producing bacteriaa BMY-28232
([Lg/ml)
Cefixime
Cefteram
Nonproducing hosts Staphylococcus aureus MS15009 Escherichia coli ML4901
0.20 0.20
3.13 0.39
0.78 0.39
Penicillinase producers Staphylococcus aureus MS15009(pI258) (2a) Escherichia coli ML4901(Rms212) (2b) Escherichia coli ML4901(Rms213) (2d) Escherichia coli ML4901(Rtel6) (2d) Escherichia coli ML4901(Rmsl49) (2c) Escherichia coli ML4901 TEM-2 (2b) Escherichia coli ML4901 SHV-1 (2b)
0.39 0.20 0.20 0.39 0.20 0.39 0.20
6.25 0.20 0.39 0.39 0.20 0.39 0.20
3.13 0.39 1.56 0.39 0.39 0.39 0.39
Cephalosporinase producers Escherichia coli ML4901(pMS161) (Enterobacter cloacae; 1) Escherichia coli ML4901(pMS182) (Proteus vulgaris; 2e) Escherichia coli ML4901(pMS185) (Citrobacterfreundii; 1) Escherichia coli ML4901(pMS500) (Escherichia coli; 1)
1.56 0.39 3.13 1.56
3.13 0.39 12.5 6.25
0.78 0.78 3.13 0.78
Cephalosporinases Escherichia coli GN14930 (chromosomal, 1) Enterobacter cloacae GN7467 (chromosomal, 1) Providencia rettgeri GN4430 (chromosomal, 1) Morganella morganii GN5307 (chromosomal, 1) Serratia marcescens GN14932 (chromosomal, 1) Pseudomonas aeruginosa GN10362 (chromosomal, 1) Proteus vulgaris GN4413 (chromosomal, 2e) Klebsiella oxytoca GN10650 (chromosomal) Pseudomonas cepacia GN11164 (chromosomal)
6.25 50 100 100 1.56 1.56
25 50 100 1.56 0.10 0.39
1.56 50 0.05 0.10 3.13 >100 3.13 3.13 6.25
a Staphylococcus aureus MS15009 and Escherichia coli ML4901 were ,3-lactamase-nonproducing host strains. Enzyme types and the classification of Bush (1) are given in parentheses.
wide range (1.56 to >100 ,uglml), as were those of cefixime and cefteram. Enterococcus faecium and Clostridium difficile were not susceptible to BMY-28232 or the other two agents. BMY-28232 had an MIC90 of 12.5 ,ug/ml against Clostridium perfringens, which was lower than those of the other two compounds tested. BMY-28232 was active against various species of gramnegative bacteria such as Escherichia coli, Shigella spp., Salmonella spp., Klebsiella spp., Proteus mirabilis, Providencia stuartii, Providencia rettgeri, Enterobacter aerogenes, Haemophilus influenzae, Branhamella catarrhalis, and Neisseria gonorrhoeae. The MIC90s against these organisms ranged from 0.025 to 1.56 uig/ml. BMY-28232 was less active against Proteus vulgaris (MIC90, 6.25 ,ug/ml) than it was against cefixime (MICg, 0.05 ,ug/ml) or cefteram (MIC90, 0.39 ,ug/ml). Against Morganella morganii, BMY28232, with an MIC90 of 12.5 ,ug/ml, showed activity comparable to those of cefixime and cefteram. Citrobacter freundii, Enterobacter cloacae, and Serratia marcescens showed broad MIC distributions against these agents, and more than 10% of the isolates were highly resistant to BMY-28232 and the other two agents. BMY-28232 had an MIC90 of 12.5 ,ug/ml against Pseudomonas cepacia and showed activity intermediate between those of cefixime and cefteram. BMY-28232 was more active against Acinetobacter calcoaceticus than it was against cefixime and cefteram. BMY-28232 had poor activity against Pseudomonas aeruginosa, Xanthomonas maltophilia, and Bacteroides fragilis (MIC90s, >100 jig/ml), which was similar to the activities of cefixime and cefteram. Antimicrobial activity against i8-lactamase-producing
strains. The MICs of BMY-28232, cefixime, and cefteram for ,B-lactamase-producing organisms are shown in Table 2. The activity of BMY-28232 against Staphylococcus aureus and Escherichia coli was not affected by the production of various types of penicillinases. The activity of BMY-28232 against an Escherichia coli strain which produced chromosomal cephalosporinases of Escherichia coli, Enterobacter cloacae, and Citrobacterfreundii was 8- to 16-fold less than that against the host strain without these enzymes. These enzymes reduced the activity of BMY-28232 to the same extent as they did the activity of cefixime. Against an Escherichia coli strain which produced Proteus vulgaris enzyme, these agents were as active as they were against the host strain. BMY-28232 was fourfold more active than cefixime and fourfold less active than cefteram against Escherichia coli GN14930. Providencia rettgeri GN4430 and Morganella morganhi GN5307 were equally susceptible to these agents. BMY-28232 was 8- to 64-fold less active than the two other agents against Serratia marcescens GN14932 and Proteus vulgaris GN4413. The activity of BMY-28232 against Klebsiella oxytoca GN10650 and Pseudomonas cepacia GN11164 was 4- to 16-fold lower than that of cefixime and was equal to or somewhat higher than that of cefteram. Enterobacter cloacae GN7467 and Pseudomonas aeruginosa GN10362 had poor susceptibilities to these agents. Hydrolysis by the I-lactamases. The relative hydrolysis rates of BMY-28232 by P-lactamases are shown in Table 3. Hydrolysis of BMY-28232 by several types of penicillinases could not be detected. Although BMY-28232, cefixime, and cefteram were poor substrates for cephalosporinases, slow hydrolysis of these agents was observed for the enzymes
BMY-28271, A NEW ORAL CEPHALOSPORIN ESTER
VOL. 34, 1990
559
TABLE 3. Hydrolysis of BMY-28232 by various 1-lactamases
E-Lactamase source Enzyme class classa Penicillinase Escherichia coli ML4901(Rms212) Escherichia coli ML4901(Rms2l3) Escherichia coli ML4901(Rtel6) Escherichia coli ML4901(Rms433) Staphylococcus aureus MS15009(pI258)
Relative hydrolysis rate
of":
BMY-28232
Cefixime
Type I (2b) Type II (2d) Type III (2d) Type IV (2c) Type V (2a)
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1990, p. 555-561
0066-48041901040555-07$02.00/0 Copyright © 1990, American Society for Microbiology
Antimicrobial Activity and Stability to 1-Lactamase of BMY-28271, a New Oral Cephalosporin Ester HIROYUKI MATSUI,'* MASAKI HIRAOKA,2 MATSUHISA INOUE,3 AND SUSUMU MITSUHASHI' Episome Institute, 2220 Kogure, Fujimi-mura, Seta-gun, Gunma 371-01,1 Preclinical Research Laboratories, Bristol-Myers Research Institute, Ltd., I Futagoyama, Sakazaki, Kota-cho, Nukata-gun, Aichi 444_0J,2 and Laboratory of Drug Resistance in Bacteria, School of Medicine, Gunma University, 39-22, Showa-machi, Maebashi, Gunma 37J,3 Japan Received 17 July 1989/Accepted 21 December 1989
BMY-28271, the acetoxyethyl ester of BMY-28232, 7-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamido]-3(Z)-propen-l-yl-3-cephem-4-carboxylic acid, is a new oral cephalosporin. BMY-28232 has a widely expanded spectrum with high activity against gram-positive and gram-negative bacteria. BMY-28232 is far more active than cefixime or cefteram against Staphylococcus aureus and Staphylococcus epidermidis. Against gram-negative bacteria, the activity of BMY-28232 was comparable to or somewhat weaker than that of cefixime or cefteram. BMY-28232 was a poor substrate for various P-lactamases. Orally administered BMY-28271 had a good therapeutic effect on systemic infections with S. aureus and some gram-negative bacteria in mice. Oral BMY-28271 was efficacious against S. aureus Smith infection: the efficacy of BMY-28271 was 80 to 90 times higher than that of cefixime or cefteram.
Many cephalosporins with broad antibacterial spectra have been used clinically for the treatment of a wide range of bacterial infections. Recently, there has been great progress in the development of oral cephalosporins, but each clinically available cephalosporin has weaknesses. Cefaclor is active against gram-positive and some gram-negative bacteria but is hydrolyzed by various f-lactamases (13). Cefixime and cefteram (T-2525) (2, 12, 14) have good activity against gram-negative bacteria and streptococci and are not hydrolyzed readily by various P-lactamases, but these compounds have relatively low activities against staphylococci (2, 6, 12, 14). BMY-28232, the parent compound of BMY-28271, is active against gram-positive and gram-negative bacteria and is relatively resistant to hydrolysis by various 1-lactamases (16). The present study deals with the in vitro antibacterial activity and P-lactamase stability of BMY-28232 and the in vivo antibacterial activity of BMY-28271 in comparison with those of cefixime and cefteram.
Determination of MICs. MICs were determined by the twofold agar dilution method. The media used for preculture and MIC determination were as follows: brain heart infusion broth (Difco Laboratories, Detroit, Mich.) and sensitivity disk agar (SDA; Nissui Seiyaku Co., Ltd., Tokyo, Japan) supplemented with 5% defibrinated horse blood for streptococci; brain heart infusion broth and SDA supplemented with 5% Fildes enrichment (Difco) for Haemophilus influenzae; sensitivity test broth (STB; Nissui Seiyaku) supplemented with 0.4% KNO3 and SDA for Pseudomonas spp.; GAM broth and agar (Nissui) for anaerobes; and STB and SDA for the other microorganisms. Organisms were precultured for 18 h at 37°C and suitably diluted in buffered saline (pH 7.0) containing 0.01% gelatin. Approximately 104 CFU of bacteria per spot were inoculated onto drug-containing agar plates with a multiloop inoculator (Microplanter; Sakuma Seisakusho Co., Ltd., Tokyo, Japan). Plates were incubated for 18 h at 37°C, except for obligate anaerobes, which were incubated for 48 h. Haemophilus influenzae and Neisseria gonorrhoeae were incubated in a candle jar, and anaerobes were incubated in an anaerobic jar. The MIC was defined as the lowest drug concentration which prevented visible growth of bacteria. Hydrolysis by 13-lactamases. The various types of P-lactamases (1, 11) used in this study were purified and stored at -80°C at the Episome Institute (3-5, 9, 18, 19). Hydrolysis of the drugs was assayed by a spectrophotometric method (17) at a substrate concentration of 100 ,uM. The susceptibility of the drugs to hydrolysis by various P-lactamases was expressed as the rate of hydrolysis relative to that of cephaloridine or benzylpenicillin, which was set at 100. For the 3-lactamases of Enterobacter cloacae GN7471 (10), Citrobacter freundii GN7391 (15), and Proteus vulgaris GN7919 (7), Km and Vmax values were determined from hydrolytic rates at various substrate concentrations by use of the Lineweaver-Burke plot. Bactericidal activity. An overnight shaking culture of organisms in STB at 37°C was inoculated into freshly prepared STB, and the culture was shaken at 37°C for 2 h. When the organisms had grown to a density of approximately 105
MATERIALS AND METHODS Antibiotics. BMY-28232 and BMY-28271 (the acetoxyethyl ester of BMY-28232) were synthesized at the Tokyo Research Center of Bristol Myers Research Institute, Ltd., Tokyo, Japan.- The other antimicrobial agents used in this study were obtained from the indicated sources: cefixime, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan; cefteram (T-2525, Ro 19-5247) and cefteram pivoxil (T-2588) (2, 12, 14), Toyama Chemical Co., Ltd., Tokyo, Japan; cefaclor, Shionogi Pharmaceutical Co., Ltd., Osaka, Japan; benzylpenicillin, Meiji Seika Kaisha, Ltd., Tokyo, Japan; cephaloridine, Nihon Glaxo Co., Ltd., Tokyo, Japan; and methicillin, Banyu Pharmaceutical Co., Ltd., Tokyo, Japan. Organisms. The bacterial strains used in this study were recent clinical isolates from various hospitals in Japan. The 3-lactamase-producing strains used in this study were maintained at the Episome Institute. All strains were stored at -800C. *
Corresponding author. 555
556
MATSUI ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. In vitro antibacterial activity of BMY-28232 against clinical isolates Organism (no. of strains)
Antibiotic
MIC Range
(4m1)a 50%
90%
Methicillin-susceptible Staphylococcus aureus (54)
BMY-28232 Cefixime Cefteram
0.39-0.78 6.25-25 3.13-12.5
0.78 12.5 6.25
0.78 25 6.25
Methicillin-susceptible Staphylococcus epidermidis (30)
BMY-28232 Cefixime Cefteram
0.025-3.13 3.13-100 0.39-12.5
0.10 6.25 1.56
0.39 50 6.25
Methicillin-resistant Staphylococcus epidermidis (39)
BMY-28232 Cefixime Cefteram
0.05->100 12.5->100 3.13->100
Streptococcus pyogenes (99)
BMY-28232 Cefixime Cefteram
100
25 100 50
1.56 0.78 0.78 >100 >100 >100 12.5 3.13 25
25 100 >100
>100 >100 >100
a 50o and 90%o, MICs for 50 and 90%o of strains tested, respectively. b Inoculum of 106 CFU per spot.
CFU/ml, antibiotics were added to the culture at concentrations ranging from one-fourth to four times the MIC determined by the agar dilution method. Samples were taken at intervals and inoculated onto drug-free heart infusion agar plates; the numbers of colonies were counted after 18 h of incubation at 37°C. Determination of oral in vivo activity. The in vivo antibacterial activities of the drugs were determined against systemic infections in mice. Ten 4-week-old male STD:ddY mice (weight, 19 to 21 g) were used for each dose of drug. An overnight culture on heart infusion agar at 37°C was suspended in physiological saline for Escherichia coli ML4707 and Klebsiella pneumoniae GN6445 or in 5% gastric mucin for Staphylococcus aureus Smith and Proteus mirabilis GN4754. A 0.5-ml portion of the bacterial suspension was inoculated intraperitoneally into each mouse. Under these conditions, all untreated mice died within 5 days. The drugs were suspended in 0.5% carboxymethyl cellulose and were given to the mice as a single oral dose immediately after infection. The 50% effective dose was calculated by the Probit method (8) from the number of mice that survived at each dose level 5 days after bacterial challenge.
RESULTS Antimicrobial activity against clinical isolates. The in vitro antimicrobial activity of BMY-28232 against gram-positive and gram-negative organisms was compared with those of cefixime and cefteram (T-2525, Ro 19-5247). Table 1 shows the concentrations of antibiotics required to inhibit the growth of 50 and 90%o (MIC%0) of the total number of strains tested. The MIC90s of BMY-28232 against gram-positive organisms such as methicillin-susceptible Staphylococcus aureus, methicillin-susceptible Staphylococcus epidermidis, Streptococcus pyogenes, and Streptococcus pneumoniae ranged from 0.025 to 0.78 ,ug/ml. BMY-28232 was 32- to 128-fold more active than cefixime and 8- to 16-fold more active than cefteram against these staphylococci. Although BMY-28232 was active against some strains of methicillinresistant Staphylococcus epidermidis, most of the methicillin-resistant staphylococci were resistant to BMY-28232 and the other two agents. BMY-28232 was active (MIC90s, 0.025 and 0.10 ,ug/ml, respectively) against Streptococcus pyogenes and Streptococcus pneumoniae. The MICs of BMY28232 against Enterococcus faecalis were distributed in a
558
MATSUI ET AL.
ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Activity of BMY-28232 against ,-lactamase producing bacteria MIC
,3-Lactamase-producing bacteriaa BMY-28232
([Lg/ml)
Cefixime
Cefteram
Nonproducing hosts Staphylococcus aureus MS15009 Escherichia coli ML4901
0.20 0.20
3.13 0.39
0.78 0.39
Penicillinase producers Staphylococcus aureus MS15009(pI258) (2a) Escherichia coli ML4901(Rms212) (2b) Escherichia coli ML4901(Rms213) (2d) Escherichia coli ML4901(Rtel6) (2d) Escherichia coli ML4901(Rmsl49) (2c) Escherichia coli ML4901 TEM-2 (2b) Escherichia coli ML4901 SHV-1 (2b)
0.39 0.20 0.20 0.39 0.20 0.39 0.20
6.25 0.20 0.39 0.39 0.20 0.39 0.20
3.13 0.39 1.56 0.39 0.39 0.39 0.39
Cephalosporinase producers Escherichia coli ML4901(pMS161) (Enterobacter cloacae; 1) Escherichia coli ML4901(pMS182) (Proteus vulgaris; 2e) Escherichia coli ML4901(pMS185) (Citrobacterfreundii; 1) Escherichia coli ML4901(pMS500) (Escherichia coli; 1)
1.56 0.39 3.13 1.56
3.13 0.39 12.5 6.25
0.78 0.78 3.13 0.78
Cephalosporinases Escherichia coli GN14930 (chromosomal, 1) Enterobacter cloacae GN7467 (chromosomal, 1) Providencia rettgeri GN4430 (chromosomal, 1) Morganella morganii GN5307 (chromosomal, 1) Serratia marcescens GN14932 (chromosomal, 1) Pseudomonas aeruginosa GN10362 (chromosomal, 1) Proteus vulgaris GN4413 (chromosomal, 2e) Klebsiella oxytoca GN10650 (chromosomal) Pseudomonas cepacia GN11164 (chromosomal)
6.25 50 100 100 1.56 1.56
25 50 100 1.56 0.10 0.39
1.56 50 0.05 0.10 3.13 >100 3.13 3.13 6.25
a Staphylococcus aureus MS15009 and Escherichia coli ML4901 were ,3-lactamase-nonproducing host strains. Enzyme types and the classification of Bush (1) are given in parentheses.
wide range (1.56 to >100 ,uglml), as were those of cefixime and cefteram. Enterococcus faecium and Clostridium difficile were not susceptible to BMY-28232 or the other two agents. BMY-28232 had an MIC90 of 12.5 ,ug/ml against Clostridium perfringens, which was lower than those of the other two compounds tested. BMY-28232 was active against various species of gramnegative bacteria such as Escherichia coli, Shigella spp., Salmonella spp., Klebsiella spp., Proteus mirabilis, Providencia stuartii, Providencia rettgeri, Enterobacter aerogenes, Haemophilus influenzae, Branhamella catarrhalis, and Neisseria gonorrhoeae. The MIC90s against these organisms ranged from 0.025 to 1.56 uig/ml. BMY-28232 was less active against Proteus vulgaris (MIC90, 6.25 ,ug/ml) than it was against cefixime (MICg, 0.05 ,ug/ml) or cefteram (MIC90, 0.39 ,ug/ml). Against Morganella morganii, BMY28232, with an MIC90 of 12.5 ,ug/ml, showed activity comparable to those of cefixime and cefteram. Citrobacter freundii, Enterobacter cloacae, and Serratia marcescens showed broad MIC distributions against these agents, and more than 10% of the isolates were highly resistant to BMY-28232 and the other two agents. BMY-28232 had an MIC90 of 12.5 ,ug/ml against Pseudomonas cepacia and showed activity intermediate between those of cefixime and cefteram. BMY-28232 was more active against Acinetobacter calcoaceticus than it was against cefixime and cefteram. BMY-28232 had poor activity against Pseudomonas aeruginosa, Xanthomonas maltophilia, and Bacteroides fragilis (MIC90s, >100 jig/ml), which was similar to the activities of cefixime and cefteram. Antimicrobial activity against i8-lactamase-producing
strains. The MICs of BMY-28232, cefixime, and cefteram for ,B-lactamase-producing organisms are shown in Table 2. The activity of BMY-28232 against Staphylococcus aureus and Escherichia coli was not affected by the production of various types of penicillinases. The activity of BMY-28232 against an Escherichia coli strain which produced chromosomal cephalosporinases of Escherichia coli, Enterobacter cloacae, and Citrobacterfreundii was 8- to 16-fold less than that against the host strain without these enzymes. These enzymes reduced the activity of BMY-28232 to the same extent as they did the activity of cefixime. Against an Escherichia coli strain which produced Proteus vulgaris enzyme, these agents were as active as they were against the host strain. BMY-28232 was fourfold more active than cefixime and fourfold less active than cefteram against Escherichia coli GN14930. Providencia rettgeri GN4430 and Morganella morganhi GN5307 were equally susceptible to these agents. BMY-28232 was 8- to 64-fold less active than the two other agents against Serratia marcescens GN14932 and Proteus vulgaris GN4413. The activity of BMY-28232 against Klebsiella oxytoca GN10650 and Pseudomonas cepacia GN11164 was 4- to 16-fold lower than that of cefixime and was equal to or somewhat higher than that of cefteram. Enterobacter cloacae GN7467 and Pseudomonas aeruginosa GN10362 had poor susceptibilities to these agents. Hydrolysis by the I-lactamases. The relative hydrolysis rates of BMY-28232 by P-lactamases are shown in Table 3. Hydrolysis of BMY-28232 by several types of penicillinases could not be detected. Although BMY-28232, cefixime, and cefteram were poor substrates for cephalosporinases, slow hydrolysis of these agents was observed for the enzymes
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TABLE 3. Hydrolysis of BMY-28232 by various 1-lactamases
E-Lactamase source Enzyme class classa Penicillinase Escherichia coli ML4901(Rms212) Escherichia coli ML4901(Rms2l3) Escherichia coli ML4901(Rtel6) Escherichia coli ML4901(Rms433) Staphylococcus aureus MS15009(pI258)
Relative hydrolysis rate
of":
BMY-28232
Cefixime
Type I (2b) Type II (2d) Type III (2d) Type IV (2c) Type V (2a)
