Affinity of Tomopenem (CS-023) for Penicillin-Binding Proteins in Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa

Abstract

Tomopenem (formerly CS-023), a novel 1β-methylcarbapenem, exhibited high affinity for penicillin-binding protein (PBP) 2 in Staphylococcus aureus, PBP 2 in Escherichia coli, and PBPs 2 and 3 in Pseudomonas aeruginosa, which are considered major lethal targets. Morphologically, tomopenem induced spherical forms in E. coli and short filamentation with bulges in P. aeruginosa, which correlated with the drug's PBP profiles. The potential of resistance of these bacteria to tomopenem was comparable to that to imipenem.

Tomopenem is a novel 1β-methylcarbapenem with a broad spectrum of activity against clinically important gram-positive and gram-negative bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (5). β-Lactams are known to bind to penicillin-binding proteins (PBPs) that function in peptidoglycan biosynthesis (16). The PBP profiles of carbapenems are consistent with their broad-spectrum activity. Moreover, their actions with certain PBPs govern the morphological changes in bacteria seen in the presence of carbapenems (2, 14). Alteration of PBPs, about which there are few reports (12) for Enterobacteriaceae and P. aeruginosa, is one of the mechanisms of carbapenem resistance. However, concern about the emergence of carbapenem resistance is growing (4). Therefore, we determined the affinity of tomopenem for PBPs in S. aureus, Escherichia coli, and P. aeruginosa, the morphological changes, and the potential for tomopenem resistance.

Membrane fractions from S. aureus ATCC 6538P, E. coli NIHJ, and P. aeruginosa ATCC 15692 were prepared as described previously (15). The drug's affinity for PBPs in S. aureus and E. coli was assayed by [¹⁴C]benzylpenicillin (Amersham Pharmacia Biotech UK Limited) (13). After contact with the gels, the imaging plate (Fuji Photo Film Co., Ltd.) was subjected to imaging analysis with a Bio-Imaging analyzer (model BAS 2000; Fuji Photo Film Co., Ltd.). The drug's affinity for PBPs in P. aeruginosa was assayed by Bocillin FL (Invitrogen Corporation) (17). Labeled PBPs were visualized by using a molecular imager FX Pro (Bio-Rad Laboratories, Inc.) and quantified by using Quantity One software (version 4.5; Bio-Rad Laboratories, Inc.). The 50% inhibitory concentrations (IC₅₀s) were calculated. The MICs and minimum bactericidal concentrations (MBCs) were measured by the standard microdilution method (10, 11). The observation of E. coli and P. aeruginosa morphology was performed by growing cultures in cation-adjusted Mueller-Hinton broth (CAMHB) and then adding tomopenem at 1/8, 1, and 8 times the MIC. After fixation, the cells were observed with a scanning electron microscope (model S-4000; Hitachi Ltd.) at a magnification of ×10,000. Spontaneous mutants of three pathogens were selected by plating overnight cultures harvested by centrifugation onto Mueller-Hinton agar containing carbapenems at 2, 4, and 8 times the MIC. Development of resistance in S. aureus and P. aeruginosa was assessed by the broth dilution method. Three independent colonies were suspended in CAMHB and CAMHB containing 0.4% (wt/vol) potassium nitrate, to give a final concentration of 5 × 10⁵ CFU/ml. After incubation in a broth containing twofold serial dilutions of carbapenems, a test tube in which colonies had grown to more than 50% of the density of the control was selected for each subsequent passage.

Tomopenem had high affinities for PBPs 1, 2, and 4 but not for PBP 3 in S. aureus (Table 1). The IC₅₀ of tomopenem for PBP 2 was fourfold higher than that of imipenem (IPM) and was similar to that of meropenem (MEM), while the MIC of tomopenem was eightfold higher than that of IPM and was similar to that of MEM. The IC₅₀s of carbapenems tested for PBP 2 seemed to be correlated with their MICs. In our previous report (6), the IC₅₀s of tomopenem for PBP 2a in two strains of MRSA were 14- to 25-fold higher than that for PBP 2, which might indicate the MICs (MIC₅₀ and MIC₉₀) of tomopenem against methicillin-sensitive S. aureus (0.12 and 0.12 μg/ml) and MRSA (4 and 8 μg/ml) (5). In E. coli, tomopenem had the highest affinity for PBP 2, as did MEM and IPM, but IPM had low affinity for PBP 3. The IC₅₀ of tomopenem for PBP 2 was threefold lower than that of IPM and was similar to that of MEM, while the MIC of tomopenem was eightfold lower than that of IPM and was similar to that of MEM. Since the synergistic inhibition of both PBP 2 and 3 was the main mechanism of the superior antibacterial activity of MEM over IPM (1), tomopenem would show superior antibacterial activity due to the inhibition of both PBP 2 and 3, just as MEM did. In P. aeruginosa, tomopenem had as high an affinity for PBP 2 as it did for PBP 3. The antibacterial activity of β-lactams is governed by the integration of the PBP affinities, the permeability of the outer membrane, the stability of β-lactamase, and the substrate extrusion rate in gram-negative bacteria. There were slight differences in the stability of β-lactamase in P. aeruginosa in response to tomopenem, IPM, and MEM (9), and the antibacterial activity of tomopenem against a MexAB-OprM-overproducing strain had less influence than that of MEM (6). Although the rate of penetration of tomopenem was not examined, the higher affinity of tomopenem for PBPs 2 and 3 compared to that of IPM and its lower recognition of efflux pumps compared to that of MEM are the main mechanisms of its better antibacterial activity.

TABLE 1.

Affinity of tomopenem, imipenem, and meropenem for pathogen PBPs

Strain	Drug	MIC (μg/ml)	IC50 (μg/ml) for target protein
			PBP 1	PBP 1a	PBP 1b	PBP 2	PBP 3	PBP 4	PBP 5	PBP 6	PBPs 5 and 6
S. aureus ATCC	Tomopenem	0.06	0.16			0.21	>32	0.094
6538P	Imipenem	0.008	0.024			0.047	0.049	0.0078
	Meropenem	0.06	0.044			0.26	>130	0.071
E. coli NIHJ	Tomopenem	0.03		1.2	1.3	0.017	0.71	>32	5.7	31
	Imipenem	0.25		0.22	1.1	0.045	19	0.44	0.34	1.0
	Meropenem	0.03		0.82	0.77	0.016	0.39	0.047	1.2	15
P. aeruginosa ATCC	Tomopenem	0.5		0.061	0.053	0.011	0.011	0.0035			5.0
15692	Imipenem	1		0.057	0.078	0.066	0.089	0.0032			0.19
	Meropenem	1		0.12	0.11	0.018	0.013	0.0033			4.5

Morphologically, in E. coli, tomopenem induced a swollen form (Fig. 1b) at 1/8 times the MIC and spherical and bulge formations (Fig. 1d) at 8 times the MIC. In P. aeruginosa, tomopenem induced filamentation (Fig. 1f) at 1/8 times the MIC and a bulge formation (Fig. 1g) at the MIC. These primary morphological responses would be in good agreement with the drug's highest affinity for PBP 2 (sphere formation) in E. coli and would be due to its simultaneously binding to PBP 2 and PBP 3 (filamentation) in P. aeruginosa.

FIG. 1.

Scanning electron micrographs of E. coli NIHJ (a to d) and P. aeruginosa ATCC 15692 (e to h) grown at 37°C for 2 h in the absence (a and e) or presence of tomopenem at 1/8 times the MIC (b and f), 1 time the MIC (c and g), and 8 times the MIC (d and h). The MICs of tomopenem against these strains are shown in Table 1. Bars represent 3 μm.

The frequencies of spontaneous resistance of S. aureus and P. aeruginosa to tomopenem were comparable to those to IPM or MEM (Table 2). The frequencies of resistance of E. coli to tomopenem and IPM were higher than that to MEM. The profiles of the susceptibility of the mutants obtained with tomopenem against S. aureus (SRR2a), E. coli (ERR2a, ERR2b, and ERR4a), and P. aeruginosa (PRR4a) showed cross-resistance to IPM and MEM, IPM, and IPM, MEM, and levofloxacin, respectively (Table 3). The reason why the frequency of spontaneous resistance of E. coli to tomopenem was higher than that to MEM is unclear, but resistance to IPM might be related to the cross-resistance to IPM against the mutants as shown by the other carbapenems. In P. aeruginosa, there is a possibility that we selected MexAB-OprM-overproducing OprD-deficient double mutants as reported previously (8), although the mechanisms of resistance were not determined.

TABLE 2.

Frequency of spontaneous mutations in S. aureus ATCC 6538P, E. coli NIHJ, and P. auruginosa ATCC 15692

Strain	Drug	MIC (μg/ml)	MBC (μg/ml)	Mutation frequency in presence of drug ata:
				2× MIC	4× MIC	8× MIC
S. aureus ATCC	Tomopenem	0.06	0.06	5.1 × 10−6	<1.0 × 10−8	<1.0 × 10−8
6538P	Imipenem	0.008	0.008	1.3 × 10−5	<1.0 × 10−8	<1.0 × 10−8
	Meropenem	0.06	0.12	1.6 × 10−5	2.7 × 10−7	<1.0 × 10−8
E. coli NIHJ	Tomopenem	0.03	0.03	1.1 × 10−5	2.0 × 10−6	1.4 × 10−7
	Imipenem	0.25	0.25	1.1 × 10−5	4.1 × 10−6	1.4 × 10−7
	Meropenem	0.03	0.03	4.3 × 10−8	<7.1 × 10−9	2.4 × 10−8
P. aeruginosa ATCC	Tomopenem	0.5	0.5	<5.6 × 10−8	5.6 × 10−9	<5.6 × 10−9
15692	Imipenem	1	2	1.1 × 10−6	<5.6 × 10−8	<5.6 × 10−9
	Meropenem	1	1	2.1 × 10−7	<5.6 × 10−9	5.6 × 10−10

^aAfter incubation for 48 h at 37°C, the colonies that grew on the plates were counted.

TABLE 3.

MICs of tomopenem, imipenem, meropenem and levofloxacin against spontaneous mutants of S. aureus, E. coli, and P. aeruginosa

Strain	Selective condition	MIC (μg/ml) (fold increase compared to parent strain MIC)
		Tomopenem	Imipenem	Meropenem	Levofloxacin
S. aureus ATCC 6538P mutants	None	0.06	0.008	0.03	0.06
SRR2a	2× MIC of tomopenem	0.25 (4)	0.03 (4)	0.25 (8)	0.12 (2)
SIR2a	2× MIC of imipenem	0.25 (4)	0.03 (4)	0.12 (4)	0.06 (1)
SMR2a	2× MIC of meropenem	0.25 (4)	0.03 (4)	0.25 (8)	0.12 (2)
SMR4a	4× MIC of meropenem	0.5 (8)	0.03 (4)	0.25 (8)	0.06 (1)
E. coli NIHJ mutants	None	0.03	0.25	0.03	0.03
ERR2a	2× MIC of tomopenem	0.12 (4)	1 (4)	0.03 (1)	0.03 (1)
ERR2b	2× MIC of tomopenem	0.12 (4)	1 (4)	0.06 (2)	0.06 (2)
ERR4a	4× MIC of tomopenem	0.12 (4)	1 (4)	0.06 (2)	0.03 (1)
ERR8a	8× MIC of tomopenem	0.12 (4)	0.5 (2)	0.06 (2)	0.03 (1)
EIR2a	2× MIC of imipenem	0.06 (2)	1 (4)	0.03 (1)	0.03 (1)
EIR2b	2× MIC of imipenem	0.06 (2)	0.5 (2)	0.03 (1)	0.03 (1)
EIR4a	4× MIC of imipenem	0.06 (2)	1 (4)	0.03 (1)	0.03 (1)
EIR4b	4× MIC of imipenem	0.06 (2)	1 (4)	0.12 (4)	0.03 (1)
EIR8a	8× MIC of imipenem	0.06 (2)	0.5 (2)	0.03 (1)	0.03 (1)
EMR2a	2× MIC of meropenem	0.06 (2)	1 (4)	0.06 (2)	0.03 (1)
P. aeruginosa ATCC 15692 mutants	None	0.25	1	0.5	0.5
PRR4a	4× MIC of tomopenem	8 (32)	8 (8)	2 (4)	2 (4)
PIR2a	2× MIC of imipenem	2 (8)	16 (16)	4 (8)	0.5 (1)
PMR2a	2× MIC of meropenem	2 (8)	16 (16)	8 (16)	2 (4)

The subculturing of S. aureus and P. aeruginosa in tomopenem led to a twofold and an eightfold decrease in susceptibility after 10 passages, respectively (Fig. 2). Since periods of extensive IPM use are associated with significant increases in resistance (3, 7), the preservation of the activity of tomopenem would require appropriate use.

FIG. 2.

Development of resistance in S. aureus ATCC 6538P (a) and P. aeruginosa ATCC 15692 (b) after serial passages. S. aureus or P. aeruginosa was cultured in a medium containing tomopenem (•), imipenem (▴), and meropenem (▪) for 18 h at 35°C. Shown are representative results with S. aureus and P. aeruginosa, since the development of resistance in all three colonies showed almost the same result with both species.

In conclusion, the PBP profiles of carbapenems tested in these pathogens were correlated with their antibacterial activities. The morphological changes induced by tomopenem corresponded to the PBP binding data. The PBP profiles of tomopenem, with potent binding to multiple essential PBPs in S. aureus, E. coli, and P. aeruginosa, are related to the broad-spectrum activities against gram-positive and gram-negative bacteria.