In Vitro Activity of S-3578, a New Broad-Spectrum Cephalosporin Active against Methicillin-Resistant Staphylococci

Antibiotics.S-3578 was synthesized in the research laboratories of Shionogi & Co., Ltd. (Osaka, Japan). Its chemical structure is shown in Fig. 1. Other compounds were purchased from commercial sources.

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FIG. 1.

Chemical structure of S-3578, 7ОІ-[2-(2-aminothiadiazol-4-yl)-2(Z)-ethoxyiminoacetamido]-3-(1-N-methylaminopropyl-1H-imidazo[4,5-b]pyridinium-4-yl)-methylcephalosporin.

Organisms.Bacterial strains used in this study were clinical isolates collected from hospitals in various parts of Japan in 2000 and 2001 and strains from our laboratory collection. S. aureus SRM710, deficient in PBP2, was used in a binding affinity assay of PBP2a of MRSA (18).

MIC determination.MICs for aerobic bacteria were determined by a microdilution broth method as recommended by the NCCLS (20) with cation-adjusted Mueller-Hinton broth (CA-MHB) (Becton Dickinson, Sparks, Md.), which was supplemented with 15 Ојg of NAD/ml, 5% yeast extract (Becton Dickinson), and 5% lysed horse blood to support the growth of Streptococcus pneumoniae and Haemophilus influenzae. CA-MHB with 2% NaCl was used to determine MICs of oxacillin for staphylococci. MICs for Neisseria gonorrhoeae were determined by an agar dilution method with GC medium (Becton Dickinson) containing growth supplement after incubation at 35В°C for 20 h in the presence of 6% CO₂. MICs for anaerobic bacteria were determined by an agar dilution method with Wilkins-Chalgren medium (Becton Dickinson) after anaerobic incubation at 35В°C for 48 h.

Determination of minimum bactericidal concentration (MBC).MBCs were determined by a macrodilution method with CA-MHB according to the NCCLS recommendations (19).

Time-kill study.The culture of a test strain at the early or mid-log phase was diluted to about 5 Г— 10⁵ CFU/ml in 5 ml of fresh CA-MHB. Antibiotics at various concentrations were added to the diluted cultures. Viable cells were counted after shaking at 37В°C for 0, 1, 2.5, 4, and 6 h. In the studies of S. aureus, the additional counting was performed at 24 h.

PBP binding affinity.The affinities of antibiotics for PBPs were determined by a competition assay with [¹⁴C]benzylpenicillin (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, England) as described previously (17, 23). The binding affinity of each compound was expressed as the concentration required to inhibit 50% of the binding of [¹⁴C]benzylpenicillin to each PBP (IC₅₀).

Population analysis and frequency of emergence of resistant colonies.An overnight culture of S. aureus in CA-MHB was diluted with fresh medium to the appropriate bacterial density and spread onto a Mueller-Hinton agar plate containing serial twofold dilutions of antibiotics. The plates were incubated for 3 days at 37В°C, and the number of colonies was counted. The frequency of emergence of resistant colonies on an agar plate containing S-3578 was shown as their ratio to the number of colonies grown on an agar plate without antibiotics.

ОІ-Lactamase stability.ОІ-Lactamases were purified partially from bacterial membrane by ion-exchange chromatography as described previously (16). The stability of antibiotics against hydrolysis by ОІ-lactamases was assessed by determining the hydrolysis rate by a spectrophotometric assay method (21). The kinetics parameters K_m and V_max were calculated from at least two independent experiments with the Michaelis-Menten formulation.

MIC determination.The antibacterial activity of S-3578 was compared with those of cefepime, ceftriaxone, ceftazidime, and imipenem. For gram-positive bacteria, vancomycin was also used as a comparator, as were oxacillin for staphylococci and penicillin G for S. pneumoniae. The results are shown in Table 1 as the MIC range, MIC₅₀ (the concentration at which the growth of 50% of the isolates was inhibited), and MIC₉₀.

S-3578 showed consistent antibacterial activity against staphylococci including methicillin-resistant strains and inhibited the growth of all clinical isolates of staphylococci at 8 Ојg/ml. The MIC₉₀s of S-3578 for MRSA and methicillin-resistant Staphylococcus epidermidis (MRSE) were 4 and 2 Ојg/ml, respectively, whereas those of other ОІ-lactam compounds were 64 Ојg/ml or more. S-3578 was fourfold less active against MRSA than was vancomycin, but its activity against MRSE was equal to that of vancomycin. For methicillin-susceptible S. aureus (MSSA) and methicillin-susceptible S. epidermidis, the MIC₉₀s of S-3578 were 2 and 0.5 Ојg/ml, respectively. It was characteristic that the MICs of S-3578 for methicillin-resistant staphylococcus strains were only two- to fourfold higher than those for methicillin-susceptible staphylococcus strains.

S-3578 had activities against penicillin-susceptible, -intermediate, and -resistant S. pneumoniae, with MIC₉₀s of 0.25, 1, and 1 Ојg/ml, respectively, which were comparable to those of ceftriaxone and cefepime. However, S-3578 was not as active against Enterococcus faecalis as were other cephalosporins, though it showed the lowest MIC₅₀ among the cephalosporins tested.

S-3578 was also active against a variety of gram-negative bacteria, although its activity was not superior to those of cefepime and imipenem, except that imipenem was less active than was S-3578 against H. influenzae. The MIC₉₀s of S-3578 for Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca were 1 Ојg/ml or less, which was comparable to those of ceftazidime but two- to eightfold higher than those of cefepime, ceftriaxone, and imipenem. Some S-3578-resistant strains of these species were also resistant to other cephalosporins. The MIC₉₀s of S-3578 for Enterobacter aerogenes, Citrobacter freundii, Serratia marcescens, Proteus mirabilis, and Providencia rettgeri ranged from 2 to 8 Ојg/ml, and most strains of Proteus vulgaris were not susceptible to S-3578. S-3578 was active against H. influenzae except for ОІ-lactamase-negative ampicillin-resistant strains with a MIC₉₀ of 0.25 Ојg/ml, regardless of the ОІ-lactamase production. Its activity was comparable to those of cefepime and ceftazidime but lower than that of ceftriaxone. The activity of S-3578 against Moraxella catarrhalis (MIC₉₀, 1 Ојg/ml) was higher than those of ceftriaxone and cefepime but fourfold lower than that of ceftazidime. S-3578 showed activity against Pseudomonas aeruginosa with a MIC₅₀ and a MIC₉₀ of 8 and 64 Ојg/ml, respectively, which were two- to fourfold higher than those of ceftazidime and cefepime. The activity of S-3578 against Acinetobacter baumannii was the highest among the cephalosporins tested, though its MIC₅₀ and MIC₉₀ were 1 and 16 Ојg/ml, respectively. S-3578, however, had the same potential against N. gonorrhoeae as did penicillin G, though other compounds were more active.

The antibacterial activities against anaerobic pathogens were determined. S-3578 inhibited growth of all strains of Peptostreptococcus spp. at 8 Ојg/ml, which meant that it was as active as ceftriaxone was. Bacteroides fragilis was not susceptible to S-3578.

Bactericidal activity.We first determined MBCs for 20 clinical isolates each of MRSA and MSSA including ОІ-lactamase producers. The MBC/MIC ratios for S-3578 against all strains were 1 or 2 (Fig. 2). In particular, the number of MSSA strains showing the ratio of 1 for S-3578 was larger than the number of those showing the same ratio for cefepime and ceftriaxone, suggesting that S-3578 was more bactericidal than the reference agents were. The production of ОІ-lactamase did not have any influence on both MBCs and MICs of S-3578 as well as of the reference compounds.

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FIG. 2.

Distribution of MBCs and MICs for 20 clinical isolates each of MRSA (A) or MSSA (B). The activity of S-3578 was compared with those of cefepime and ceftriaxone against MSSA. Open and closed circles indicate ОІ-lactamase-producing and nonproducing strains, respectively.

Next, we constructed the killing kinetics curves for MRSA and MSSA strains. S-3578 caused a time-dependent decrease of viable cells of the MSSA strain Smith at one or more times the MIC until 6 h, as did cefepime and ceftriaxone (Fig. 3B). Furthermore, the number of viable cells after 24-h exposure to S-3578 was significantly lower than the number after exposure to the reference compounds. S-3578 also showed similar bactericidal activity against the MRSA strain SR3637 at two or more times the MIC (Fig. 3A). Moreover, the decreases of viable cells with 10 clinical isolates each of MRSA or MSSA were compared after 24-h exposure to twice the MIC of each antibiotic. S-3578 caused more than a 4-log₁₀-CFU/ml reduction on the average for both MRSA and MSSA, while cefepime and ceftriaxone demonstrated only less than a 2-log₁₀-CFU/ml reduction for MSSA (Fig. 4). These results indicated that the bactericidal activity of S-3578 was more potent than those of cefepime and ceftriaxone against S. aureus, irrespective of methicillin resistance.

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FIG. 3.

Killing kinetics curves for S-3578 and reference compounds against S. aureus SR3637 (MRSA) (A) and Smith (MSSA) (B).

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FIG. 4.

Decrease of viable cells in 10 clinical isolates each of MRSA or MSSA after 24-h exposure to twice the MIC of S-3578. Cefepime and ceftriaxone were used as comparators for MSSA. An open circle indicates decrease of viable cell counts of each strain compared to the initial inoculum, and a bar indicates the average of those of 10 strains.

The MBCs of S-3578 for 20 clinical isolates of E. coli for which MICs were 0.13 to 1 Ојg/ml ranged from 0.25 to 1 Ојg/ml, while the MBCs and MICs of cefepime ranged from 0.25 to 2 Ојg/ml and from 0.13 to 2 Ојg/ml, respectively (Fig. 5). All strains tested were ОІ-lactamase producers, indicating that the activity of S-3578 was not affected by ОІ-lactamase. Furthermore, the killing kinetics curves for E. coli NIHJ JC-2 and P. aeruginosa SR24706 indicated that S-3578 also had time-dependent bactericidal activity against these species which was comparable to those of the reference compounds (Fig. 6).

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FIG. 5.

Distribution of MBCs and MICs for 20 clinical isolates of E. coli. The activity of S-3578 was compared with that of cefepime. All strains produced ОІ-lactamases.

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FIG. 6.

Killing kinetics curves for S-3578 and reference compounds against E. coli NIHJ JC-2 (A) and P. aeruginosa SR24706 (B).

Binding affinity for PBPs.S-3578 was found to have high affinity for PBP2a of MRSA, with an IC₅₀ of 4.5 Ојg/ml (Table 2). This characteristic seemed to reflect the anti-MRSA activity of S-3578. In contrast, cefepime, which was not active against MRSA, had a very low affinity for PBP2a. S-3578 also showed high affinities for PBP1 and -2 of S. aureus with IC₅₀s of less than 0.5 Ојg/ml, as did ceftriaxone and cefepime.

Cefepime had high affinities for PBP2 and -3 of E. coli with IC₅₀s of less than 1 Ојg/ml, and ceftriaxone had affinities for PBP1A, -1B, -2, and -3 (Table 3). On the other hand, S-3578 had high affinity only for PBP3. Although the IC₅₀s of S-3578 for PBP1A, -3, and -4 of P. aeruginosa ranged from 0.31 to 0.43 Ојg/ml (Table 4), its binding affinity was not as high as those of cefepime and ceftazidime, the IC₅₀s of which for PBP3 were 10-fold less than that of S-3578.

Frequency of emergence of resistant colonies.In order to estimate the frequency of resistance development, we first carried out population analysis on two clinical isolates of MRSA, the low-level-methicillin-resistant strain SR19754 and the high-level-methicillin-resistant strain SR19760. As shown in Fig. 7, strain SR19754 had a subpopulation resistant to imipenem at a frequency of about 10^в€’6 in spite of the low MIC of imipenem for this strain, that is, it exhibited a heteroresistance profile. In contrast, the viabilities of both strains against S-3578 sharply decreased with increase of the antibiotic concentration. In particular, it was notable that no colonies emerged on agar plates containing twice the MIC of S-3578. Furthermore, about 10⁹ bacterial cells each of 20 clinical isolates of MRSA were spread on agar plates containing S-3578 and incubated for 3 days at 35В°C. For all isolates, no colonies appeared on the plates at twice the MIC, and emergence from an isolate for which the MIC was 8 Ојg/ml did not occur even at the MIC (data not shown). From these results, the frequency at which resistant colonies emerged was estimated to be below 10^в€’9.

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FIG. 7.

Population analysis profiles of two clinical isolates of MRSA. Strains SR19754 (A) and SR19760 (B) are low-level- and high-level-methicillin-resistant strains, respectively. The MICs of S-3578 and imipenem for each strain are indicated in the figure. Closed circles and open triangles indicate viable cell counts on plates containing S-3578 or imipenem, respectively.

ОІ-Lactamase stability.The hydrolysis of S-3578 and cefepime by a ОІ-lactamase purified from S. aureus SR5644 was not detected at 100 Ојg of the compounds per ml (data not shown), indicating that S-3578 was stable against hydrolysis by the ОІ-lactamase from S. aureus. S-3578, as well as cefepime, was stable against TEM-1 and a class A ОІ-lactamase from K. pneumoniae GN69 (22) because their hydrolysis rates were less than 0.1% of that of ampicillin at the substrate concentration of 100 Ојg/ml (data not shown). Although the K_m values of S-3578 against various AmpC enzymes from C. freundii, Enterobacter cloacae, and P. aeruginosa were lower than those of cefepime, its relative V_max/K_m values were comparable to those of cefepime. These results indicated that S-3578 had higher affinity for AmpC enzymes but was as stable as cefepime was against hydrolysis (Table 5). On the other hand, S-3578 was not as stable as cefepime was against a Toho-1-like extended-spectrum ОІ-lactamase and a class B IMP-1-like enzyme (data not shown).