621
DIAGN MICROBIOLINFECT DIS 1992;15:621-625
Antibacterial Oxazolidinones In vitro Activity of a N e w Analogue, E3709 William Brumfitt and Jeremy M.T. Hamilton-Miller
The oxazolidinone compound E3709, which contains a 4-pyridyl group, was found to be more active in vitro than other members of this series, such as DuP 721. MIC9ofor staphylococci(including methicillin-resistant isolates), streptococci (including Enterococcus faecalis), Clostridia, and diphtheroids was 1000 i~g/ml. At a concentration of 10 i~g/ml, E3709 was bactericidal for selected Grampositive species. A postantibiotic effect of 3 hr was observed against staphylococci. Resistance to E3709 was not detected.
INTRODUCTION
now been synthesized, which differs from earlier members in that it has a 4-pyridyl grouping in the side chain (Figure 1). As this is ionizable, the properties of E3709 both in vitro and in vivo might be expected to be substantially different from those of the oxazolidinone antibiotics so far tested. We report here an assessment of the in vitro antibacterial properties of E3709.
There is a need for new agents with activity against Gram-positive bacteria, especially those that are resistant to many conventional antibiotics, such as the enterococci and methicillin-resistant Staphylococcus aureus (MRSA). Therefore, the oxazolidinone series, developed in the laboratories of DuPont de Nemours and Company (Gregory et al., 1990) is of great potential importance, as its unique chemical structure makes the possibility of cross-resistance very unlikely. Representatives of the series tested so far include S-6123 (Daly et al., 1988b), DuP 105, and DuP 721 (Slee et al., 1987; Neu et al., 1988; Daly et al., 1988a; Brumfitt and Hamilton-Miller, 1988; Mini et al., 1989). These three compounds show varying degrees of activity of a bacteriostatic nature against Gram-positive bacteria and a small number of Gramnegative species, such as Haemophilus influenzae and Bacteroides spp. The mode of action is unique among antibacterial compounds, as it involves the inhibition of a very early step in protein synthesis (Eustice et al., 1988). An additional compound in this series, E3709, has From the Department of Medical Microbiology,The Royal Free Hospital School of Medicine, London, UK. Address reprint requests to Dr. J.M.T. Hamilton-Miller, Department of Medical Microbiology,The Royal Free Hospital, Pond Street, London NW3 2QG, UK. Received 27 August 1991, revised and accepted 4 December 1991. © 1992Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$5.00
MATERIALS A N D METHODS Antimicrobial Agents The hydrochloride of E3709 (given the code number XA043), which is water soluble, was supplied by DuPont (Wilmington, DE, USA). Comparator antibiotics were cefaclor (Eli Lilly, Basingstoke, UK), tetracycline hydrochloride (Pfizer, Sandwich, England, UK), metronidazole (May and Baker, Dagenham, UK), and vancomycin hydrochloride (Sigma Chemical Company, St. Louis, MO, USA) as appropriate. Corrections were made for stated potencies.
Media IsoSensitest agar (ISA), IsoSensitest broth (ISB), and brain-heart infusion broth (BHI) were purchased from Oxoid (Basingstoke, UK). Robertson's cooked meat medium (RCM) was from Southern Group Laboratories (London SE, UK). When streptococci (except group-D strains) were being tested, 5% lysed horse
622
W. Brumfitt and J.M.T. Hamilton-Miller
0
"\ / \ NHCOCH 3
Compound
X
DuP 105
CH3SO
DuP 721
CH3CO
E 3709
FIGURE 1
Structures of oxazolidinone antibacterials.
diluted culture and the 1:100 dilution in water, 1-~1 volumes were inoculated to the surface of plates of ISA containing doubling dilutions of the appropriate antibiotics. Inoculum sizes were thus 106 and 10 4 colony-forming units (CFU). Plates were incubated for 18 hr at 37°C in air or in anaerobic jars using the GASPAK system (as appropriate) and minimum inhibitory concentration (MIC) was read. Plates that had been inoculated with 106 CFU were then replicated using Dralon pads to plates of fresh ISA. The latter were incubated for 18 hr in the appropriate atmosphere and the minimum bactericidal concentration (MBC) was read. Killing of at least 99.9% of the original inoculum was indicated by growth of no more than 10 colonies on replicate plates, as the plating efficacy from Dralon pads is 1%. The above procedure was carried out for the strains listed in Tables 1 and 2. For the other strains that were considerably less sensitive to E3709, concentrations of 10, 100, and 1000 }xg/ml only were tested, and no MBC determinations were made.
Time-Kill Experiments blood were added to the agar, and for diphtheroids, media were supplemented with horse serum (0.1 ml added to 3 ml broth, and 1% added to solid media).
Bacterial Strains The following were tested: 214 strains of Gram-positive species, consisting of Staphylococcus aureus, S.
epidermidis, S. saprophyticus, Streptococcus pyogenes, St. agalactiae, Enterococcus faecalis, St. pneumoniae, viridans-group streptococci, diphtheroids, and Clostridium spp.; 87 strains of Gram-negative species, namely, Bacteroides fragilis, Haemophilus influenzae, Moraxella catarrhalis, Acinetobacter spp., Citrobacter freundii, Providencia rettgeri, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Enterobacter cloacae, and Pseudomonas aeruginosa; and five strains of Candida albicans. Further details as to numbers are given in Tables 1 and 2. MRSA strains were taken at random from our International Collection (Maple et al., 1989). The viridans-group streptococci, diphtheroids, and M. catarrhalis were normal flora from the throats or nares of laboratory staff or patients at the Royal Free Hospital. Other than these, all strains had been isolated within the previous 3 years from infections occurring in patients at or attending the Royal Free Hospital.
E3709 was added to five 500-ml conical flasks, each containing 100 ml ISB inoculated with 5 x 107 CFU to give concentrations in the range 1-16 times the MIC; a sixth flask was a control. Flasks were shaken in an Orbital incubator at 100 rpm at 37°C, and samples were taken for viable counting at 0, 2, 4, 6, 8, and 24 hr.
Postantibiotic Effect Bacteria (5 x 105 CFU/ml, in 100 ml ISB contained in a 500-ml conical flask) were exposed to various concentrations of E3709 (usually ranging from I x to 8 x MIC) at 37°C, shaking at 100 rpm. After 1 hr, samples were diluted 1:100 in fresh ISB, a viable count was made at once, and these cultures were shaken at 37°C. Over the course of the next 4 hr, viable counts were made every 30 rain. A control flask to which no antibiotic had been added was treated in an identical way. Plots of viable count against time were made, and the postantibiotic effect (PAE) was calculated as the time required for count in antibiotic-exposed culture to increase tenfold (1 log) above the count immediately after the dilution step, minus the corresponding figure for the control culture.
Selection of Resistance Determination of M i n i m u m Inhibitory and Bactericidal Concentrations Basic media were supplemented as described below. Strains to be tested were cultured overnight in BHI (aerobes) or RCM (anaerobes) at 37°C. Of the un-
The gradient plate technique, as described by us previously (Brumfitt and Hamilton-Miller, 1988), was used. A gradient of 0-20 p,g/ml E3709 was made in ISA, and five strains each of MRSA, S. aureus, methicillin-resistant S. epidermidis, and methicillin-sensi-
623
Antibacterial Oxazolidinone
TABLE 1
In vitro Activity of E3709 Against Gram-Positive Bacteria
Bacterial Species
No. of Strains Tested
MIC (~g/ml) 104 CFU Antimicrobial Agent
Range
50%
90%
Staphylococcus aureus (MR)
20
E3709 Vancomycin
0.13-0.25 0.5-2
0.25 1
S. aureus (MS)
20
E3709 Cefaclor Tetracycline
0.13-0.5 0.25-1 0.13-128
0.25 0.25 0.5
S. epidermidis (MR)
20
E3709 Vancomycin
0.13-0.25 0.5-2
0.13 2
0.25 2
S. epidermidis (MS)
20
E3709 Cefaclor
0.06-0.25 0.06-4
0.06 0.13
0.13 2
S. saprophyticus
19
E3709 Cefaclor Tetracycline
0.13-0.5 0.6-2 0.6-32
0.25 1 0.5
St. pyogenes
16
E3709 Cefaclor Tetracycline
St. agalactiae
15
E3709 Cefaclor Tetracycline
0.13-0.25 0.5-1 0.5-128 0.13-2 0.25--1
Enterococci
20
E3709 Ampicillin
Viridans-group streptococcus
14
E3709 Cefaclor Tetracycline
Pneumococci
12
Diphtheroids a
Clostridium spp.
DIAGN MICROBIOLINFECT DIS 1992;15:621-625
Antibacterial Oxazolidinones In vitro Activity of a N e w Analogue, E3709 William Brumfitt and Jeremy M.T. Hamilton-Miller
The oxazolidinone compound E3709, which contains a 4-pyridyl group, was found to be more active in vitro than other members of this series, such as DuP 721. MIC9ofor staphylococci(including methicillin-resistant isolates), streptococci (including Enterococcus faecalis), Clostridia, and diphtheroids was 1000 i~g/ml. At a concentration of 10 i~g/ml, E3709 was bactericidal for selected Grampositive species. A postantibiotic effect of 3 hr was observed against staphylococci. Resistance to E3709 was not detected.
INTRODUCTION
now been synthesized, which differs from earlier members in that it has a 4-pyridyl grouping in the side chain (Figure 1). As this is ionizable, the properties of E3709 both in vitro and in vivo might be expected to be substantially different from those of the oxazolidinone antibiotics so far tested. We report here an assessment of the in vitro antibacterial properties of E3709.
There is a need for new agents with activity against Gram-positive bacteria, especially those that are resistant to many conventional antibiotics, such as the enterococci and methicillin-resistant Staphylococcus aureus (MRSA). Therefore, the oxazolidinone series, developed in the laboratories of DuPont de Nemours and Company (Gregory et al., 1990) is of great potential importance, as its unique chemical structure makes the possibility of cross-resistance very unlikely. Representatives of the series tested so far include S-6123 (Daly et al., 1988b), DuP 105, and DuP 721 (Slee et al., 1987; Neu et al., 1988; Daly et al., 1988a; Brumfitt and Hamilton-Miller, 1988; Mini et al., 1989). These three compounds show varying degrees of activity of a bacteriostatic nature against Gram-positive bacteria and a small number of Gramnegative species, such as Haemophilus influenzae and Bacteroides spp. The mode of action is unique among antibacterial compounds, as it involves the inhibition of a very early step in protein synthesis (Eustice et al., 1988). An additional compound in this series, E3709, has From the Department of Medical Microbiology,The Royal Free Hospital School of Medicine, London, UK. Address reprint requests to Dr. J.M.T. Hamilton-Miller, Department of Medical Microbiology,The Royal Free Hospital, Pond Street, London NW3 2QG, UK. Received 27 August 1991, revised and accepted 4 December 1991. © 1992Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$5.00
MATERIALS A N D METHODS Antimicrobial Agents The hydrochloride of E3709 (given the code number XA043), which is water soluble, was supplied by DuPont (Wilmington, DE, USA). Comparator antibiotics were cefaclor (Eli Lilly, Basingstoke, UK), tetracycline hydrochloride (Pfizer, Sandwich, England, UK), metronidazole (May and Baker, Dagenham, UK), and vancomycin hydrochloride (Sigma Chemical Company, St. Louis, MO, USA) as appropriate. Corrections were made for stated potencies.
Media IsoSensitest agar (ISA), IsoSensitest broth (ISB), and brain-heart infusion broth (BHI) were purchased from Oxoid (Basingstoke, UK). Robertson's cooked meat medium (RCM) was from Southern Group Laboratories (London SE, UK). When streptococci (except group-D strains) were being tested, 5% lysed horse
622
W. Brumfitt and J.M.T. Hamilton-Miller
0
"\ / \ NHCOCH 3
Compound
X
DuP 105
CH3SO
DuP 721
CH3CO
E 3709
FIGURE 1
Structures of oxazolidinone antibacterials.
diluted culture and the 1:100 dilution in water, 1-~1 volumes were inoculated to the surface of plates of ISA containing doubling dilutions of the appropriate antibiotics. Inoculum sizes were thus 106 and 10 4 colony-forming units (CFU). Plates were incubated for 18 hr at 37°C in air or in anaerobic jars using the GASPAK system (as appropriate) and minimum inhibitory concentration (MIC) was read. Plates that had been inoculated with 106 CFU were then replicated using Dralon pads to plates of fresh ISA. The latter were incubated for 18 hr in the appropriate atmosphere and the minimum bactericidal concentration (MBC) was read. Killing of at least 99.9% of the original inoculum was indicated by growth of no more than 10 colonies on replicate plates, as the plating efficacy from Dralon pads is 1%. The above procedure was carried out for the strains listed in Tables 1 and 2. For the other strains that were considerably less sensitive to E3709, concentrations of 10, 100, and 1000 }xg/ml only were tested, and no MBC determinations were made.
Time-Kill Experiments blood were added to the agar, and for diphtheroids, media were supplemented with horse serum (0.1 ml added to 3 ml broth, and 1% added to solid media).
Bacterial Strains The following were tested: 214 strains of Gram-positive species, consisting of Staphylococcus aureus, S.
epidermidis, S. saprophyticus, Streptococcus pyogenes, St. agalactiae, Enterococcus faecalis, St. pneumoniae, viridans-group streptococci, diphtheroids, and Clostridium spp.; 87 strains of Gram-negative species, namely, Bacteroides fragilis, Haemophilus influenzae, Moraxella catarrhalis, Acinetobacter spp., Citrobacter freundii, Providencia rettgeri, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Enterobacter cloacae, and Pseudomonas aeruginosa; and five strains of Candida albicans. Further details as to numbers are given in Tables 1 and 2. MRSA strains were taken at random from our International Collection (Maple et al., 1989). The viridans-group streptococci, diphtheroids, and M. catarrhalis were normal flora from the throats or nares of laboratory staff or patients at the Royal Free Hospital. Other than these, all strains had been isolated within the previous 3 years from infections occurring in patients at or attending the Royal Free Hospital.
E3709 was added to five 500-ml conical flasks, each containing 100 ml ISB inoculated with 5 x 107 CFU to give concentrations in the range 1-16 times the MIC; a sixth flask was a control. Flasks were shaken in an Orbital incubator at 100 rpm at 37°C, and samples were taken for viable counting at 0, 2, 4, 6, 8, and 24 hr.
Postantibiotic Effect Bacteria (5 x 105 CFU/ml, in 100 ml ISB contained in a 500-ml conical flask) were exposed to various concentrations of E3709 (usually ranging from I x to 8 x MIC) at 37°C, shaking at 100 rpm. After 1 hr, samples were diluted 1:100 in fresh ISB, a viable count was made at once, and these cultures were shaken at 37°C. Over the course of the next 4 hr, viable counts were made every 30 rain. A control flask to which no antibiotic had been added was treated in an identical way. Plots of viable count against time were made, and the postantibiotic effect (PAE) was calculated as the time required for count in antibiotic-exposed culture to increase tenfold (1 log) above the count immediately after the dilution step, minus the corresponding figure for the control culture.
Selection of Resistance Determination of M i n i m u m Inhibitory and Bactericidal Concentrations Basic media were supplemented as described below. Strains to be tested were cultured overnight in BHI (aerobes) or RCM (anaerobes) at 37°C. Of the un-
The gradient plate technique, as described by us previously (Brumfitt and Hamilton-Miller, 1988), was used. A gradient of 0-20 p,g/ml E3709 was made in ISA, and five strains each of MRSA, S. aureus, methicillin-resistant S. epidermidis, and methicillin-sensi-
623
Antibacterial Oxazolidinone
TABLE 1
In vitro Activity of E3709 Against Gram-Positive Bacteria
Bacterial Species
No. of Strains Tested
MIC (~g/ml) 104 CFU Antimicrobial Agent
Range
50%
90%
Staphylococcus aureus (MR)
20
E3709 Vancomycin
0.13-0.25 0.5-2
0.25 1
S. aureus (MS)
20
E3709 Cefaclor Tetracycline
0.13-0.5 0.25-1 0.13-128
0.25 0.25 0.5
S. epidermidis (MR)
20
E3709 Vancomycin
0.13-0.25 0.5-2
0.13 2
0.25 2
S. epidermidis (MS)
20
E3709 Cefaclor
0.06-0.25 0.06-4
0.06 0.13
0.13 2
S. saprophyticus
19
E3709 Cefaclor Tetracycline
0.13-0.5 0.6-2 0.6-32
0.25 1 0.5
St. pyogenes
16
E3709 Cefaclor Tetracycline
St. agalactiae
15
E3709 Cefaclor Tetracycline
0.13-0.25 0.5-1 0.5-128 0.13-2 0.25--1
Enterococci
20
E3709 Ampicillin
Viridans-group streptococcus
14
E3709 Cefaclor Tetracycline
Pneumococci
12
Diphtheroids a
Clostridium spp.
