ANTIMICROBIAL AGENTS
AND
CHEMOTHERAPY, Feb. 1991,
p.
329-334
Vol. 35, No. 2
0066-4804191/020329-06$02.00/0 Copyright
)
1991, American Society for Microbiology
In Vitro Activity of a Catechol-Substituted Cephalosporin, GR69153 Department
R. WISE,* J. M. ANDREWS, J. P. ASHBY, AND D. THORNBER of Medical Microbiology, Dudley Road Hospital, Birmingham, United Kingdom Received 26 June 1990/Accepted 21 November 1990
The in vitro activity of GR69153, a new catechol-substituted cephalosporin, was compared with those of ceftazidime, imipenem, meropenem, and ceftriaxone against 604 recent clinical isolates and other strains with known mechanisms of resistance. The MICs of GR69153 for 90% of the members of the family Enterobacteriaceae tested were less than 0.5 ,ug/mI, with the exceptions of those for Serratia spp. (4 ,ug/ml), Citrobacter spp. (2 jig/ml), and Enterobacter spp. (8 jig/mI). Ninety percent of Pseudomonas aeruginosa isolates were susceptible to s1 ,ug of GR69153 per ml. With the exception of methicillin-resistant strains, 90% of Staphylococcus aureus isolates were susceptible to c2 jig/ml, and GR69153 was four- to eightfold more active than ceftazidime and ceftriaxone against these strains. Isolates of Haemophilus influenzae, Branhamella catarrhalis, Neisseria spp., and Streptococcus pneumoniae (penicillin susceptible) were highly susceptible (MIC for 90% of the strains, s0.12 jIg/ml). GR69153 was stable to hydrolysis by the TEM-1 and TEM-5, SHV-1 and SHV-2, and Kl ,I-lactamases, but some susceptibility to hydrolysis by the TEM-3, TEM-9, and P99 enzymes was observed. The protein-binding activity of GR69153 was 74.5 to 66.8%, depending on the concentration, and serum had little effect upon activity.
A number of catechol-substituted penicillins and cephalosporins which show enhanced activity against members of the family Enterobacteriaceae and Pseudomonas aeruginosa have been synthesized (3, 9, 14). It is postulated that they owe this activity to a siderophore transport route (3) controlled by the tonB gene. GR69153 is a new catechol-substituted cephalosporin with an extended serum elimination half-life (12) and has the formula (6R, 7R, 2-Z, S)-7-{2-(2 aminothiazol-4-yl)-2-
yield a viable count of about 109 CFU/ml. Streptococci, enterococci, H. influenzae, and Neisseria spp. were grown in brain heart infusion broth (Oxoid Ltd., Basingstoke, England) plus 20 ,ug of NAD per ml (Sigma Chemicals, Poole, England). Bacteroides spp. were grown in WilkinsChalgren broth (Oxoid Ltd.) plus 0.25% sodium succinate. Clostridia were grown in Wilkins-Chalgren broth supplemented with 1% Tween 80 (which had previously been shown to enhance growth). The viable counts were comparable in each broth. One microliter of a 1:1,000 dilution of an overnight culture or, for a few selected strains for which increased inoculum sizes were to be studied, a 1:10 diluted inoculum was transferred to the surface of the antibiotic-containing agar by a multipoint inoculating device (Denley-Tech, Billingshurst, England). The final sizes of inocula on the plates were therefore 104 and 106 CFU. The medium used for the agar dilution procedure was Iso-Sensitest agar (pH 7.2; Oxoid) supplemented with 5% horse blood plus 20 ,ug of NAD per ml to support growth of streptococci, H. influenzae, and Neisseria spp.; for anaerobes, Wilkins-Chalgren agar was used. All plates were incubated in air at 37°C for 24 h, except that the anaerobes were grown in an anaerobic cabinet in an atmosphere of 10% hydrogen-10% carbon dioxide-80% nitrogen and the H. influenzae and Neisseria spp. were incubated in air enriched with 6% carbon dioxide. In addition, Staphylococcus aureus was also incubated in air at 30°C, with 5% sodium chloride added to the medium. The MIC of the antibiotic was defined as that concentration at which no more than two colonies were detected. With the larger inoculum size, a slight haze of growth was ignored. The effects of human serum on the MIC and MBC of GR69153 were studied with 10 strains (two each of Klebsiella spp., Escherichia coli, P. aeruginosa, S. pneumoniae, and Staphylococcus aureus) by a method based on that of Pearson et al. (10); the bactericidal endpoint was 99.9% lethality. An overnight broth culture of these organisms was inoculated into 1 ml of Iso-Sensitest broth with 20 and 70% human serum and decreasing concentrations of the antimito
[carboxy-(3,4-dihydroxyphenyl)methoxyimino]}acetamidoceph-3-em-carboxylic acid. Preliminary studies suggest that both in vitro and in vivo activities against members of the family Enterobacteriaceae and P. aeruginosa are high (the MICs for the latter ranging from 0.03 to 2 ,ug/ml) (5). In this study, we have investigated the in vitro activity of this agent against a wide range of pathogenic bacteria. Comparative agents which also exhibited a broad spectrum of antibacterial activity were chosen.
MATERIALS AND METHODS A total of 611 strains were tested, of which 604 were recent clinical isolates from different patients. The remainder were well-characterized resistant strains. The antibiotics and their sources were as follows: GR69153, cephaloridine, and ceftazidime, Glaxo Research Laboratories, Greenford, England; cefotaxime, Roussel Laboratories, Denham, England; imipenem, M.S.D. Hoddesdon, Herts, England; meropenem, Imperial Chemical Industries, Macclesfield, England; ceftriaxone, Roche Products, Welwyn Garden City, England; and amoxycillin, Beecham Research Laboratories, Brentford, England. Susceptibility testing. The susceptibilities of the strains were studied by using a routine agar plate dilution method. The inocula were prepared as follows. For all strains except streptococci (including Streptococcus pneumoniae), enterococci, Neisseria spp., Haemophilus influenzae, and anaerobes, the organisms were grown overnight in nutrient broth *
Corresponding author.
crobial agent. 329
330
WISE ET AL.
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Activity of GR69153 compared with those of other agents
Organism (no. of isolates)
Escherichia coli (50)
Antibiotic
GR69153
Ceftazidime
Imipenem Ceftriaxone Meropenem Klebsiella spp. (49)
GR69153
Ceftazidime
Imipenem Ceftriaxone Meropenem Proteus mirabilis (50)
GR69153
Ceftazidime
Imipenem Meropenem
Ceftriaxone Proteus vulgaris (22)
GR69153 Ceftazidime
Imipenem Meropenem Ceftriaxone Morganella morganii (21)
GR69153 Ceftazidime
Imipenem
Ceftriaxone
Meropenem Providencia spp. (19)
GR69153 Ceftazidime
Imipenem
Ceftriaxone
Meropenem Serratia spp. (20)
GR69153 Ceftazidime
Imipenem Ceftriaxone
Meropenem Citrobacter spp. (10)
GR69153 Ceftazidime
Imipenem Ceftriaxone
Meropenem Enterobacter spp. (10)
GR69153 Ceftazidime
Imipenem Ceftriaxone
Meropenem Pseudomonas aeruginosa (36)
GR69153 Ceftazidime
Imipenem Ceftriaxone Meropenem
Staphylococcus aureus (35) (including 5 methicillin-resistant strains)
GR69153 Ceftazidime
Imipenem Ceftriaxone
Meropenem Methicillin
509% 0.03 0.06 0.06 0.06 0.008
90%0 0.06 0.12 0.12 0.06 0.008
0.03 0.06 0.12 0.03 0.015
0.12 0.12 0.25 0.12 0.015
0.008-4 0.008-0.03
0.015 0.06 1 0.008 0.06
0.06 0.06 2 0.008 0.12
0.015-0.12 0.03-0.06 0.25-2 0.008 0.03-0.25
0.12 0.06 1 0.03 0.03
0.12 0.06 2 0.12 0.12
0.015-0.25 0.06 0.12-4 0.008-0.5 0.03-0.12
0.12 0.06 1 0.008 0.03
0.25 0.12 1 0.03 0.03
0.015-4 0.008-16 0.25-2 0.008-16 0.015-0.06
0.03 0.12 0.5 0.015 0.03
0.03 0.25 1 0.06 0.12
0.015-8 0.06-8 0.06-4
0.5 0.25 0.5 0.12 0.03
4 1 0.5 16 0.06
0.25 0.25 0.25 0.12 0.015
2 2 1 8 0.12
0.25 0.25 0.5 0.12 0.03
8 8 4 64 2
0.5 1 1 8 0.5
1 8 2 32 2
2 8 0.015 8 0.06 2
Range 0.03-0.5 0.015-0.5
0.06-0.25 0.03-0.12
0.004-0.015 0.015-1 0.015-1
0.06-0.5
0.008-64 0.015-2
0.03-8 0.06-8 0.06-4
0.008-64 0.015-2 0.12-8 0.06-8
0.06-4
0.06-64 0.015-2 0.12-8 0.06-8 0.06-4
0.06-64
0.015-2
0.25-64 0.25-64 0.5-8
4->128
0.12-8
16 0.12->128 32 2-128 0.06 0.015-0.12 16 0.03-64 0.25 0.03-0.25 32 1-32 Continued on following page
VOL. 35, 1991
IN VITRO ACTIVITY OF GR69153
331
TABLE 1-Continued Organism (no. of isolates)
Antibiotic
50%o Staphylococcus epidermidis (18)
GR69153 Ceftazidime
Imipenem Meropenem Ceftriaxone
Methicillin
Staphylococcus saprophyticus (20)
GR69153 Ceftazidime
Imipenem Ceftriaxone Meropenem
Methicillin
Enterococcus faecalis (10)
GR69153 Ceftazidime
Imipenem Ceftriaxone Meropenem Streptococcus pneumoniae (19)
GR69153 Ceftazidime
Haemophilus influenzae (34) (including 9 3-lactamase producers)
0.06
4
8 >128 1 16 2
64 >128 1 128 4
0.03-0.25 1-8
0.015->128
0.25-64 0.06->128 0.03-2
0.015-0.12 0.5-32
4-128
32->128 0.5-1
4-128 2-4
0.03
0.06
0.12 0.12 0.06 0.5 0.004
0.008-2 0.002-0.008
0.03
0.002-0.3
0.06 0.06
0.008-0.12 Q.004-0.06 0.002-0.015
Imipenem Meropenem
0.03 0.06 0.004
GR69153 Ceftazidime
Imipenem Ceftriaxone Meropenem
0.008 0.03 0.03 0.002 0.008
GR69153 Ceftazidime
0.06
GR69153 Ceftazidime
Imipenem Ceftriaxone Meropenem
The protein-binding activity of GR69153 in human serum was estimated in triplicate by an ultrafiltration technique with a Centriflo cone (Amicon Corp., Lexington, Mass.) with an exclusion limit of 50,000 Da. The concentrations of GR69153 used were 5, 50, and 100 ,ug/ml. The ultrafiltrate was assayed by a microbiological method against standards prepared in phosphate buffer at pH 7.0 (the pH of the ultrafiltrate). The indicator organism was E. coli NCTC 10418, and the medium was Oxoid antibiotic medium no. 1. P-Lactamase stability. The enzymes studied are shown in Table 4. Cell-free sonic extracts from the strains containing the single characterized enzymes were prepared from midexponential-phase cultures in tryptone soya broth as previ-
4 16 0.03 32 0.25 8
0.015-0.03 0.25-8
GR69153 Ceftazidime
Imipenem Meropenem
a 50%1 and 90%, MIC for 50 and 90% of the isolates, respectively.
2 8 0.03 8
Range 0.062 2-8
Imipenem Meropenem
Ceftriaxone Bacteroides spp. (23)
0.5 2 0.03 8 0.25 4
2 4 0.06 0.25 0.12
Ceftriaxone Neisseria gonorrhoeae (23) (including 2 ,-Iactamase producers)
0.12 2 0.015 1 0.06 2
0.12 0.25 0.004 0.015 0.008
Ceftriaxone
Branhamella sp. (30) (including 11 13-lactamase producers)
90%
0.015 0.25 0.004
0.06 8 32 0.06 8 0.12
0.008 0.008 0.03 0.12 0.5 0.004 0.12 >128 >128 0.25 >128 0.25
0.015-2 0.12-4 0.002-0.12
0.004-0.25 0.002-0.12 0.008-0.25 0.03-0.12
0.008-0.12
0.002-0.015 0.015-0.03 0.03-0.12 0.12-0.5 0.002-0.008
0.03-0.12 0.25->28
4->128
0.03-64 1->128
0.06->128
ously described (1, 4). The extracts were assayed for protein by the method of Lowry et al. (7), and isoelectric focusing was carried out on LKB ampholine polyacrylamide gel plates with a LKB Multiphor II electrophoretic unit with a constant 2.5-kV power supply. Nitrocefin was used to detect ,-lactamase activity. Hydrolysis studies were carried out for amoxycillin, cephaloridine, ceftazidime, cefotaxime, imipenem, meropenem, and GR69153 with a temperature-controlled UV spectrophotometer (Perkin Elmer Lambda 2; Perkin Elmer, Beaconsfield, England) at 37°C. All antibiotic solutions were prepared immediately before use in 50 mM sodium phosphate buffer, pH 7, and the reactions were monitored at those
332
WISE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. MICs against known P-lactamase-producing strains Strain
Enterobacter cloacae Klebsiella aerogenes Escherichia coli E. coli E. coli E. coli E. coli a
l3-Lactamasea
MIC (Lg/ml)
GR69153 Imipenem Ceftriaxone
P99 (GP1) 128 Kl (GP4) 0.25 TEM-3 (GP3) 1 TEM-5 (GP3) 1 TEM-9 (GP3) 0.5 OXA-2 (GP5) 0.03 OXA-3 (GPS) 0.03
0.12 0.12 0.12 0.06 0.12 0.12 0.12
64 8 8 8 4 0.03 0.03
GP, Richmond and Sykes group.
wavelengths which gave the maximum changes in absorbance before and after hydrolysis. The spectral parameters (in nanometers) for the drugs used in this study were as follows: cephaloridine, 260; ceftazidime and cefotaxime, 255; amoxycillin, 250; imipenem and meropenem, 298; and GR69153, 242. The reported kinetic constants were derived by the halftime analysis of single reaction progress curves (8). For slowly hydrolyzed compounds, affinities were determined by the competing-substrate approach, and the best estimates of Vm were calculated from initial-rate experiments (6, 8). The relative V.max rates were compared with that of cephaloridine, which was taken to be 100%. The value calculated for Vmax1Km can be taken as a measure of the efficiency of hydrolysis as defined by Pollock (11). Induction studies. Induction of ,-lactamase activity in four strains (one each of Providencia stuartii, Citrobacterfreundii, Enterobacter cloacae, and P. aeruginosa) was measured. The log-phase cultures of the strains were exposed to concentrations equivalent to one-half of the MICs of cefoxitin, piperacillin, and GR69153 for 4 h (15). Enzyme activity was measured spectrophotometrically by using nitrocefin as the substrate and was standardized against total protein. RESULTS The activity of GR69153 is shown in Table 1 and compared with those of the other agents. Against members of the family Enterobacteriaceae, GR69153 displayed activity very similar to those of ceftazidime, ceftriaxone, and imipenem, with meropenem being -2-fold more active. Exceptions to this were the poor
activity of imipenem against the Proteus spp., Morganella morganii, and Providencia spp. and the lesser activity of ceftriaxone against Serratia and Enterobacter spp. Certain strains of M. morganii and Citrobacter, Enterobacter, Serratia, and Providencia spp. all showed decreased susceptibilities to GR69153 (1 MIC < 8 jg/ml), and the same strains had decreased susceptibilities to ceftriaxone (2 c MIC 64 ,ug/ml). Table 2 shows the activities of GR69153, imipenem, and ceftriaxone (as representative of the cephalosporins) against known ,-lactamase-producing strains. This might suggest that ceftriaxone is more readily hydrolyzed by the TEM series enzymes than GR69153 and that imipenem is remarkably stable. It is also possible that there are differences in cell permeability which might explain these -
-
differences. In general, GR69153 was twofold more active than ceftazidime against P. aeruginosa, although 5 of 36 strains tested were 8- to 16-fold more susceptible to the new agent; there therefore appeared to be two populations with susceptibility to ceftazidime (modal MICs, 1 and 8 ,ug/ml for 25 of 36 and 11 of 36 strains, respectively) yet only one population with susceptibility to GR69153. One clinical isolate was inhibited by 64 ,ug each of GR69153 and ceftazidime per ml, by 128 ,ug of ceftriaxone per ml, and by 2 and 8 ,ug of imipenem and meropenem per ml, respectively. With the exception of methicillin-resistant strains, Staphylococcus aureus strains were susceptible to 128 pug/ml. Staphylococcus epidermidis strains were markedly more susceptible to GR69153 (MIC for 90% of the strains, 0.5 p.g/ml) than Staphylococcus saprophyticus strains (MIC for 90% of the strains, 4 ,ug/ml). A similar interspecies difference was seen for the other cephalosporins, except for imipenem and meropenem. H. influenzae and Neisseria gonorrhoeae strains were susceptible to all the agents studied. The 1-lactamase producers of both genera were as susceptible as the nonproducers.
The strains of Branhamella catarrhalis were all susceptible to -0.25 ,ug of GR69153 per ml; however, two populations existed, one for which the modal MIC was 0.008 ,g/ml and one for which the modal MIC was 0.12 ,g/ml. Two such populations were also seen among the strains susceptible to ceftriaxone (modal MICs, 0.015 and 0.5 ,g/ml) but not
TABLE 3. Effects of serum on MIC and MBC of GR69153 Concn (>g/ml)
Organism (no. of isolates)
Escherichia coli (431) Escherichia coli (428) Klebsiella spp. (284) Klebsiella spp. (200) Pseudomonas aeruginosa (58) Pseudomonas aeruginosa (304) Staphylococcus aureus (408) Staphylococcus aureus (407) Streptococcus pneumoniae (846) Streptococcus pneumoniae (847) a NVG, No visible growth in broth.
20%'o Serum
Iso-Sensitest broth
70o Serum
MIC
MBC
MIC
MBC
MICa
MBC
0.12 0.12 0.03 0.03 1 1 0.5 0.25 0.12 0.12
0.12 0.25 0.06 0.12 4 2 1 1 0.12 0.12
0.03 0.06 0.03 0.03 0.03 0.03 1 2 0.25 0.5
0.03 0.12 0.06 0.06 2 1 1 4 0.25 0.5
0.03 0.06 NVG 0.03 NVG NVG 2 4 0.25 2
0.06 0.12
0.03 0.03 0.5 0.5 4 8 0.25 2
VOL. 35, 1991
IN VITRO ACTIVITY OF GR69153
333
TABLE 4. Relative efficiency of hydrolysisa Vmax/Kmax for the following drug:
J3-Lactamase
TEM-1 TEM-3 TEM-5 TEM-9 SHV-1 SHV-2 K-1 P99
Amoxycillin
Ceftazidime
Cefotaxime
Imipenem
Meropenem
GR69153
1,786 170 1,483 1,085 609 52 399 0.03
AND
CHEMOTHERAPY, Feb. 1991,
p.
329-334
Vol. 35, No. 2
0066-4804191/020329-06$02.00/0 Copyright
)
1991, American Society for Microbiology
In Vitro Activity of a Catechol-Substituted Cephalosporin, GR69153 Department
R. WISE,* J. M. ANDREWS, J. P. ASHBY, AND D. THORNBER of Medical Microbiology, Dudley Road Hospital, Birmingham, United Kingdom Received 26 June 1990/Accepted 21 November 1990
The in vitro activity of GR69153, a new catechol-substituted cephalosporin, was compared with those of ceftazidime, imipenem, meropenem, and ceftriaxone against 604 recent clinical isolates and other strains with known mechanisms of resistance. The MICs of GR69153 for 90% of the members of the family Enterobacteriaceae tested were less than 0.5 ,ug/mI, with the exceptions of those for Serratia spp. (4 ,ug/ml), Citrobacter spp. (2 jig/ml), and Enterobacter spp. (8 jig/mI). Ninety percent of Pseudomonas aeruginosa isolates were susceptible to s1 ,ug of GR69153 per ml. With the exception of methicillin-resistant strains, 90% of Staphylococcus aureus isolates were susceptible to c2 jig/ml, and GR69153 was four- to eightfold more active than ceftazidime and ceftriaxone against these strains. Isolates of Haemophilus influenzae, Branhamella catarrhalis, Neisseria spp., and Streptococcus pneumoniae (penicillin susceptible) were highly susceptible (MIC for 90% of the strains, s0.12 jIg/ml). GR69153 was stable to hydrolysis by the TEM-1 and TEM-5, SHV-1 and SHV-2, and Kl ,I-lactamases, but some susceptibility to hydrolysis by the TEM-3, TEM-9, and P99 enzymes was observed. The protein-binding activity of GR69153 was 74.5 to 66.8%, depending on the concentration, and serum had little effect upon activity.
A number of catechol-substituted penicillins and cephalosporins which show enhanced activity against members of the family Enterobacteriaceae and Pseudomonas aeruginosa have been synthesized (3, 9, 14). It is postulated that they owe this activity to a siderophore transport route (3) controlled by the tonB gene. GR69153 is a new catechol-substituted cephalosporin with an extended serum elimination half-life (12) and has the formula (6R, 7R, 2-Z, S)-7-{2-(2 aminothiazol-4-yl)-2-
yield a viable count of about 109 CFU/ml. Streptococci, enterococci, H. influenzae, and Neisseria spp. were grown in brain heart infusion broth (Oxoid Ltd., Basingstoke, England) plus 20 ,ug of NAD per ml (Sigma Chemicals, Poole, England). Bacteroides spp. were grown in WilkinsChalgren broth (Oxoid Ltd.) plus 0.25% sodium succinate. Clostridia were grown in Wilkins-Chalgren broth supplemented with 1% Tween 80 (which had previously been shown to enhance growth). The viable counts were comparable in each broth. One microliter of a 1:1,000 dilution of an overnight culture or, for a few selected strains for which increased inoculum sizes were to be studied, a 1:10 diluted inoculum was transferred to the surface of the antibiotic-containing agar by a multipoint inoculating device (Denley-Tech, Billingshurst, England). The final sizes of inocula on the plates were therefore 104 and 106 CFU. The medium used for the agar dilution procedure was Iso-Sensitest agar (pH 7.2; Oxoid) supplemented with 5% horse blood plus 20 ,ug of NAD per ml to support growth of streptococci, H. influenzae, and Neisseria spp.; for anaerobes, Wilkins-Chalgren agar was used. All plates were incubated in air at 37°C for 24 h, except that the anaerobes were grown in an anaerobic cabinet in an atmosphere of 10% hydrogen-10% carbon dioxide-80% nitrogen and the H. influenzae and Neisseria spp. were incubated in air enriched with 6% carbon dioxide. In addition, Staphylococcus aureus was also incubated in air at 30°C, with 5% sodium chloride added to the medium. The MIC of the antibiotic was defined as that concentration at which no more than two colonies were detected. With the larger inoculum size, a slight haze of growth was ignored. The effects of human serum on the MIC and MBC of GR69153 were studied with 10 strains (two each of Klebsiella spp., Escherichia coli, P. aeruginosa, S. pneumoniae, and Staphylococcus aureus) by a method based on that of Pearson et al. (10); the bactericidal endpoint was 99.9% lethality. An overnight broth culture of these organisms was inoculated into 1 ml of Iso-Sensitest broth with 20 and 70% human serum and decreasing concentrations of the antimito
[carboxy-(3,4-dihydroxyphenyl)methoxyimino]}acetamidoceph-3-em-carboxylic acid. Preliminary studies suggest that both in vitro and in vivo activities against members of the family Enterobacteriaceae and P. aeruginosa are high (the MICs for the latter ranging from 0.03 to 2 ,ug/ml) (5). In this study, we have investigated the in vitro activity of this agent against a wide range of pathogenic bacteria. Comparative agents which also exhibited a broad spectrum of antibacterial activity were chosen.
MATERIALS AND METHODS A total of 611 strains were tested, of which 604 were recent clinical isolates from different patients. The remainder were well-characterized resistant strains. The antibiotics and their sources were as follows: GR69153, cephaloridine, and ceftazidime, Glaxo Research Laboratories, Greenford, England; cefotaxime, Roussel Laboratories, Denham, England; imipenem, M.S.D. Hoddesdon, Herts, England; meropenem, Imperial Chemical Industries, Macclesfield, England; ceftriaxone, Roche Products, Welwyn Garden City, England; and amoxycillin, Beecham Research Laboratories, Brentford, England. Susceptibility testing. The susceptibilities of the strains were studied by using a routine agar plate dilution method. The inocula were prepared as follows. For all strains except streptococci (including Streptococcus pneumoniae), enterococci, Neisseria spp., Haemophilus influenzae, and anaerobes, the organisms were grown overnight in nutrient broth *
Corresponding author.
crobial agent. 329
330
WISE ET AL.
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Activity of GR69153 compared with those of other agents
Organism (no. of isolates)
Escherichia coli (50)
Antibiotic
GR69153
Ceftazidime
Imipenem Ceftriaxone Meropenem Klebsiella spp. (49)
GR69153
Ceftazidime
Imipenem Ceftriaxone Meropenem Proteus mirabilis (50)
GR69153
Ceftazidime
Imipenem Meropenem
Ceftriaxone Proteus vulgaris (22)
GR69153 Ceftazidime
Imipenem Meropenem Ceftriaxone Morganella morganii (21)
GR69153 Ceftazidime
Imipenem
Ceftriaxone
Meropenem Providencia spp. (19)
GR69153 Ceftazidime
Imipenem
Ceftriaxone
Meropenem Serratia spp. (20)
GR69153 Ceftazidime
Imipenem Ceftriaxone
Meropenem Citrobacter spp. (10)
GR69153 Ceftazidime
Imipenem Ceftriaxone
Meropenem Enterobacter spp. (10)
GR69153 Ceftazidime
Imipenem Ceftriaxone
Meropenem Pseudomonas aeruginosa (36)
GR69153 Ceftazidime
Imipenem Ceftriaxone Meropenem
Staphylococcus aureus (35) (including 5 methicillin-resistant strains)
GR69153 Ceftazidime
Imipenem Ceftriaxone
Meropenem Methicillin
509% 0.03 0.06 0.06 0.06 0.008
90%0 0.06 0.12 0.12 0.06 0.008
0.03 0.06 0.12 0.03 0.015
0.12 0.12 0.25 0.12 0.015
0.008-4 0.008-0.03
0.015 0.06 1 0.008 0.06
0.06 0.06 2 0.008 0.12
0.015-0.12 0.03-0.06 0.25-2 0.008 0.03-0.25
0.12 0.06 1 0.03 0.03
0.12 0.06 2 0.12 0.12
0.015-0.25 0.06 0.12-4 0.008-0.5 0.03-0.12
0.12 0.06 1 0.008 0.03
0.25 0.12 1 0.03 0.03
0.015-4 0.008-16 0.25-2 0.008-16 0.015-0.06
0.03 0.12 0.5 0.015 0.03
0.03 0.25 1 0.06 0.12
0.015-8 0.06-8 0.06-4
0.5 0.25 0.5 0.12 0.03
4 1 0.5 16 0.06
0.25 0.25 0.25 0.12 0.015
2 2 1 8 0.12
0.25 0.25 0.5 0.12 0.03
8 8 4 64 2
0.5 1 1 8 0.5
1 8 2 32 2
2 8 0.015 8 0.06 2
Range 0.03-0.5 0.015-0.5
0.06-0.25 0.03-0.12
0.004-0.015 0.015-1 0.015-1
0.06-0.5
0.008-64 0.015-2
0.03-8 0.06-8 0.06-4
0.008-64 0.015-2 0.12-8 0.06-8
0.06-4
0.06-64 0.015-2 0.12-8 0.06-8 0.06-4
0.06-64
0.015-2
0.25-64 0.25-64 0.5-8
4->128
0.12-8
16 0.12->128 32 2-128 0.06 0.015-0.12 16 0.03-64 0.25 0.03-0.25 32 1-32 Continued on following page
VOL. 35, 1991
IN VITRO ACTIVITY OF GR69153
331
TABLE 1-Continued Organism (no. of isolates)
Antibiotic
50%o Staphylococcus epidermidis (18)
GR69153 Ceftazidime
Imipenem Meropenem Ceftriaxone
Methicillin
Staphylococcus saprophyticus (20)
GR69153 Ceftazidime
Imipenem Ceftriaxone Meropenem
Methicillin
Enterococcus faecalis (10)
GR69153 Ceftazidime
Imipenem Ceftriaxone Meropenem Streptococcus pneumoniae (19)
GR69153 Ceftazidime
Haemophilus influenzae (34) (including 9 3-lactamase producers)
0.06
4
8 >128 1 16 2
64 >128 1 128 4
0.03-0.25 1-8
0.015->128
0.25-64 0.06->128 0.03-2
0.015-0.12 0.5-32
4-128
32->128 0.5-1
4-128 2-4
0.03
0.06
0.12 0.12 0.06 0.5 0.004
0.008-2 0.002-0.008
0.03
0.002-0.3
0.06 0.06
0.008-0.12 Q.004-0.06 0.002-0.015
Imipenem Meropenem
0.03 0.06 0.004
GR69153 Ceftazidime
Imipenem Ceftriaxone Meropenem
0.008 0.03 0.03 0.002 0.008
GR69153 Ceftazidime
0.06
GR69153 Ceftazidime
Imipenem Ceftriaxone Meropenem
The protein-binding activity of GR69153 in human serum was estimated in triplicate by an ultrafiltration technique with a Centriflo cone (Amicon Corp., Lexington, Mass.) with an exclusion limit of 50,000 Da. The concentrations of GR69153 used were 5, 50, and 100 ,ug/ml. The ultrafiltrate was assayed by a microbiological method against standards prepared in phosphate buffer at pH 7.0 (the pH of the ultrafiltrate). The indicator organism was E. coli NCTC 10418, and the medium was Oxoid antibiotic medium no. 1. P-Lactamase stability. The enzymes studied are shown in Table 4. Cell-free sonic extracts from the strains containing the single characterized enzymes were prepared from midexponential-phase cultures in tryptone soya broth as previ-
4 16 0.03 32 0.25 8
0.015-0.03 0.25-8
GR69153 Ceftazidime
Imipenem Meropenem
a 50%1 and 90%, MIC for 50 and 90% of the isolates, respectively.
2 8 0.03 8
Range 0.062 2-8
Imipenem Meropenem
Ceftriaxone Bacteroides spp. (23)
0.5 2 0.03 8 0.25 4
2 4 0.06 0.25 0.12
Ceftriaxone Neisseria gonorrhoeae (23) (including 2 ,-Iactamase producers)
0.12 2 0.015 1 0.06 2
0.12 0.25 0.004 0.015 0.008
Ceftriaxone
Branhamella sp. (30) (including 11 13-lactamase producers)
90%
0.015 0.25 0.004
0.06 8 32 0.06 8 0.12
0.008 0.008 0.03 0.12 0.5 0.004 0.12 >128 >128 0.25 >128 0.25
0.015-2 0.12-4 0.002-0.12
0.004-0.25 0.002-0.12 0.008-0.25 0.03-0.12
0.008-0.12
0.002-0.015 0.015-0.03 0.03-0.12 0.12-0.5 0.002-0.008
0.03-0.12 0.25->28
4->128
0.03-64 1->128
0.06->128
ously described (1, 4). The extracts were assayed for protein by the method of Lowry et al. (7), and isoelectric focusing was carried out on LKB ampholine polyacrylamide gel plates with a LKB Multiphor II electrophoretic unit with a constant 2.5-kV power supply. Nitrocefin was used to detect ,-lactamase activity. Hydrolysis studies were carried out for amoxycillin, cephaloridine, ceftazidime, cefotaxime, imipenem, meropenem, and GR69153 with a temperature-controlled UV spectrophotometer (Perkin Elmer Lambda 2; Perkin Elmer, Beaconsfield, England) at 37°C. All antibiotic solutions were prepared immediately before use in 50 mM sodium phosphate buffer, pH 7, and the reactions were monitored at those
332
WISE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. MICs against known P-lactamase-producing strains Strain
Enterobacter cloacae Klebsiella aerogenes Escherichia coli E. coli E. coli E. coli E. coli a
l3-Lactamasea
MIC (Lg/ml)
GR69153 Imipenem Ceftriaxone
P99 (GP1) 128 Kl (GP4) 0.25 TEM-3 (GP3) 1 TEM-5 (GP3) 1 TEM-9 (GP3) 0.5 OXA-2 (GP5) 0.03 OXA-3 (GPS) 0.03
0.12 0.12 0.12 0.06 0.12 0.12 0.12
64 8 8 8 4 0.03 0.03
GP, Richmond and Sykes group.
wavelengths which gave the maximum changes in absorbance before and after hydrolysis. The spectral parameters (in nanometers) for the drugs used in this study were as follows: cephaloridine, 260; ceftazidime and cefotaxime, 255; amoxycillin, 250; imipenem and meropenem, 298; and GR69153, 242. The reported kinetic constants were derived by the halftime analysis of single reaction progress curves (8). For slowly hydrolyzed compounds, affinities were determined by the competing-substrate approach, and the best estimates of Vm were calculated from initial-rate experiments (6, 8). The relative V.max rates were compared with that of cephaloridine, which was taken to be 100%. The value calculated for Vmax1Km can be taken as a measure of the efficiency of hydrolysis as defined by Pollock (11). Induction studies. Induction of ,-lactamase activity in four strains (one each of Providencia stuartii, Citrobacterfreundii, Enterobacter cloacae, and P. aeruginosa) was measured. The log-phase cultures of the strains were exposed to concentrations equivalent to one-half of the MICs of cefoxitin, piperacillin, and GR69153 for 4 h (15). Enzyme activity was measured spectrophotometrically by using nitrocefin as the substrate and was standardized against total protein. RESULTS The activity of GR69153 is shown in Table 1 and compared with those of the other agents. Against members of the family Enterobacteriaceae, GR69153 displayed activity very similar to those of ceftazidime, ceftriaxone, and imipenem, with meropenem being -2-fold more active. Exceptions to this were the poor
activity of imipenem against the Proteus spp., Morganella morganii, and Providencia spp. and the lesser activity of ceftriaxone against Serratia and Enterobacter spp. Certain strains of M. morganii and Citrobacter, Enterobacter, Serratia, and Providencia spp. all showed decreased susceptibilities to GR69153 (1 MIC < 8 jg/ml), and the same strains had decreased susceptibilities to ceftriaxone (2 c MIC 64 ,ug/ml). Table 2 shows the activities of GR69153, imipenem, and ceftriaxone (as representative of the cephalosporins) against known ,-lactamase-producing strains. This might suggest that ceftriaxone is more readily hydrolyzed by the TEM series enzymes than GR69153 and that imipenem is remarkably stable. It is also possible that there are differences in cell permeability which might explain these -
-
differences. In general, GR69153 was twofold more active than ceftazidime against P. aeruginosa, although 5 of 36 strains tested were 8- to 16-fold more susceptible to the new agent; there therefore appeared to be two populations with susceptibility to ceftazidime (modal MICs, 1 and 8 ,ug/ml for 25 of 36 and 11 of 36 strains, respectively) yet only one population with susceptibility to GR69153. One clinical isolate was inhibited by 64 ,ug each of GR69153 and ceftazidime per ml, by 128 ,ug of ceftriaxone per ml, and by 2 and 8 ,ug of imipenem and meropenem per ml, respectively. With the exception of methicillin-resistant strains, Staphylococcus aureus strains were susceptible to 128 pug/ml. Staphylococcus epidermidis strains were markedly more susceptible to GR69153 (MIC for 90% of the strains, 0.5 p.g/ml) than Staphylococcus saprophyticus strains (MIC for 90% of the strains, 4 ,ug/ml). A similar interspecies difference was seen for the other cephalosporins, except for imipenem and meropenem. H. influenzae and Neisseria gonorrhoeae strains were susceptible to all the agents studied. The 1-lactamase producers of both genera were as susceptible as the nonproducers.
The strains of Branhamella catarrhalis were all susceptible to -0.25 ,ug of GR69153 per ml; however, two populations existed, one for which the modal MIC was 0.008 ,g/ml and one for which the modal MIC was 0.12 ,g/ml. Two such populations were also seen among the strains susceptible to ceftriaxone (modal MICs, 0.015 and 0.5 ,g/ml) but not
TABLE 3. Effects of serum on MIC and MBC of GR69153 Concn (>g/ml)
Organism (no. of isolates)
Escherichia coli (431) Escherichia coli (428) Klebsiella spp. (284) Klebsiella spp. (200) Pseudomonas aeruginosa (58) Pseudomonas aeruginosa (304) Staphylococcus aureus (408) Staphylococcus aureus (407) Streptococcus pneumoniae (846) Streptococcus pneumoniae (847) a NVG, No visible growth in broth.
20%'o Serum
Iso-Sensitest broth
70o Serum
MIC
MBC
MIC
MBC
MICa
MBC
0.12 0.12 0.03 0.03 1 1 0.5 0.25 0.12 0.12
0.12 0.25 0.06 0.12 4 2 1 1 0.12 0.12
0.03 0.06 0.03 0.03 0.03 0.03 1 2 0.25 0.5
0.03 0.12 0.06 0.06 2 1 1 4 0.25 0.5
0.03 0.06 NVG 0.03 NVG NVG 2 4 0.25 2
0.06 0.12
0.03 0.03 0.5 0.5 4 8 0.25 2
VOL. 35, 1991
IN VITRO ACTIVITY OF GR69153
333
TABLE 4. Relative efficiency of hydrolysisa Vmax/Kmax for the following drug:
J3-Lactamase
TEM-1 TEM-3 TEM-5 TEM-9 SHV-1 SHV-2 K-1 P99
Amoxycillin
Ceftazidime
Cefotaxime
Imipenem
Meropenem
GR69153
1,786 170 1,483 1,085 609 52 399 0.03
