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Ritipenem (FCE 22101), a penem antibiotic, is potent against both gram-positive and -negative bacteria, and its acetoxymethyl ester (FCE 22891; ritipenem-acoxil) is orally available (4, 17). Haemophilus influenzae is an important human pathogen that causes severe respiratory infections, otitis, and meningitis (9). Ritipenem has strong activity against -lactamase-producing ampicillin-resistant H. influenzae, as well as susceptible strains (3, 6), because of its stability against TEM-1 -lactamase, which is produced by resistant strains. The occurrence of resistant strains has increased to up to 20% of the total clinical isolates in some areas (1, 8).

There have been many reports on the activity of ritipenem against many kinds of bacterial pathogens (3, 4, 6, 10, 17), but detailed analyses of its bactericidal activity against H. influenzae and its affinities for penicillin-binding proteins (PBPs) have not yet been performed. It has been demonstrated in Escherichia coli that bacterial lysis induced by a -lactam antibiotic is associated with binding affinities for some PBPs (5, 14, 18); however, correlations between the lysis of H. influenzae cells and affinities for PBPs have not been well understood. In this study, we investigated bacteriolytic activities against H. influenzae and the PBP-binding affinities of ritipenem and some characteristic -lactams.

The ampicillin-susceptible strain H. influenzae IID983, which was provided by the Institute of Medical Science, University of Tokyo, was used for all examinations. Ritipenem was kindly provided by Pharmacia and Upjohn (Milan, Italy). The following -lactam antibiotics were purchased from their respective manufacturers: amoxicillin, cefotaxime, and cefsulodin (Sigma Chemical Co., St. Louis, Mo.); aztreonam (Eisai Co., Ltd., Tokyo, Japan); cefdinir (Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan); fropenem (Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan); and meropenem (Sankyo Co., Ltd., Tokyo, Japan).

The MICs of these -lactams were determined by the agar dilution method. The cells, grown in Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) supplemented with 15 µg each of hemin (Sigma) and -NAD (Nacalai Tesque, Kyoto, Japan) per ml (2), were diluted with Mueller-Hinton broth to 5 × 10⁶ CFU/ml to prepare the inoculum. The cell suspension (5 µl) was spotted onto chocolate agar plates, which were prepared from Mueller-Hinton agar (Difco) containing twofold serial dilutions of each antibiotic, by a multipoint inoculator (Sakuma, Tokyo, Japan). MICs were determined after incubation for 18 h at 37°C (Table 1).

View this table: [in this window] [in a new window]   TABLE 1.   Affinities of ritipenem and other -lactams for PBPs of H. influenzae IID983

Time-kill and lytic studies were performed at the MIC and four times the MIC of each antibiotic. Cells grown in Mueller-Hinton broth supplemented with 15 µg each of hemin and -NAD per ml were prepared in a 5-ml culture (1.2 × 10⁶ CFU/ml) with fresh broth. The cultures were incubated with continuous shaking in a water bath at 37°C. After a 1-h incubation, each antibiotic was added to a culture. At selected times, 30 µl of each culture was serially diluted with saline and plated onto chocolate agar in order to determine the viable cell count (CFU/milliliter). The culture turbidity was monitored by recording the optical density at 620 nm with a spectrophotometer (Spectronic 301; Milton Roy Company, Rochester, N.Y.).

The affinities of ritipenem and the selected -lactams for PBPs in cell membrane fractions of the strain were determined by a competitive assay with ³H-labeled benzylpenicillin (³H-PCG is [phenyl-4(n)-³H]benzylpenicillin [37 MBq/ml] plus 20 µg of PCG/ml; Amersham, Little Chalfont, Buckinghamshire, United Kingdom), according to the methods of Spratt (11) and Tuomanen et al. (14). PBPs labeled with ³H-PCG were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using a running gel consisting of 12.5% acrylamide (GIBCO-BRL, Life Technologies Inc., Gaithersburg, Md.) and 0.083% N,N-methylenebisacrylamide (GIBCO-BRL). After electrophoresis, fractionated PBPs were electrically transferred to a nitrocellulose membrane (Immobirone P^SQ; Millipore Corporation, Bedford, Mass.) under a constant current of 150 mA for 50 min by a semidry-blotting apparatus (Nihon Eido, Tokyo, Japan), using a buffer consisting of 50 mM Tris base, 200 mM glycine, and 20% methanol. The transferred proteins on the membrane were fixed with methanol-acetic acid (ratio, 6/1) and then rinsed with methanol. The membrane was dried and attached to a phosphorimaging plate of a BAS-2000 image-analyzing system (Fujifilm, Tokyo, Japan) at room temperature for 3 days. The radioactivity of the PBPs on the plate was scanned and analyzed by the image-analyzing system. The affinity of a -lactam for a PBP was expressed as the concentration of the -lactam needed to cause a 50% inhibition of ³H-PCG binding (IC₅₀).

Figure 1A and B show the bacterial cell counts and turbidities of the cultures treated with the eight selected -lactams at their MICs and at four times their MICs, respectively. The bacterial cell counts in both cultures treated with ritipenem at its MIC and four times its MIC decreased to 2.0 × 10³ CFU/ml at 8 h, the lowest count obtained with the tested -lactams. The culture turbidities decreased rapidly after treatments with ritipenem and cefsulodin at four times their MICs, and complete lysis was observed at 4 h. The bactericidal activity of cefdinir was similar to those of fropenem, amoxicillin, and cefsulodin. Moderate decreases in the culture turbidities were observed after treatments with cefdinir and fropenem at four times their MICs, but these changes were not so obvious in the cultures treated with these drugs at their MICs. The turbidities of the cultures treated with cefotaxime, aztreonam, meropenem, and amoxicillin at their MICs and four times their MICs increased gradually, and they did not decrease much after treatments at four times their MICs, despite the evident decreases in the cell counts. We observed the cells treated with these -lactams at their MICs for 2 h with a differential interference-contrast microscope (NTF2; Nikon, Tokyo, Japan). Bacterial debris were predominantly observed in the cultures treated with ritipenem (Fig. 2B) and cefsulodin (Fig. 2E). Treatment with fropenem at its MIC resulted in the formation of ovoid cells without lysis (Fig. 2C), but the cells became round and partially lysed after a treatment at four times the MIC. Cefdinir at its MIC induced the formation of filamentous cells without any obvious lysis (Fig. 2D), but it caused lysis without filamentation at four times the MIC as well as ritipenem did. Lysed cells were not observed after treatments with cefotaxime, aztreonam, meropenem, and amoxicillin at their MICs (Fig. 2F to I, respectively) or at four times their MICs.

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Bactericidal and bacteriolytic activities of ritipenem (), fropenem (), cefdinir (), cefsulodin (), cefotaxime (), aztreonam (), meropenem (), and amoxicillin () at their MICs (A) and four times their MICs (B) against H. influenzae IID983. Solid and dotted lines without symbols show the viable-cell counts and turbidity of control cultures, respectively. Cells were incubated in supplemented Mueller-Hinton medium, and each drug was added at time zero. O.D., optical density.

View larger version (97K): [in this window] [in a new window]   FIG. 2.   Photomicrographs of H. influenzae IID983 cells treated with one antibiotic at its MIC for 2 h, as follows: none (A), ritipenem (B), fropenem (C), cefdinir (D), cefsulodin (E), cefotaxime (F), aztreonam (G), meropenem (H), and amoxicillin (I). Magnification, ×1,000.

We next examined the affinities of these -lactams for PBPs. Figure 3 shows the binding profiles of ritipenem and cefsulodin for some PBPs, and Table 1 shows the IC₅₀s of all eight tested -lactams. The competitive assay with ³H-PCG revealed eight PBPs of H. influenzae IID983, and they were determined to be PBPs 1a, 1b, 2, 3a, 3b, 4, 5, and 6 on the basis of a previous report (2). Ritipenem showed a high affinity for PBP 1b, followed by PBPs 2 and 1a. The IC₅₀ for PBP 1b was 0.04 times the MIC. The affinities for PBPs 3a and 3b were much lower than that for PBP 1b, and the IC₅₀s for PBPs 3a and 3b were 100- and 26-fold higher than that for PBP 1b (4.6 and 1.2 times the MIC), respectively. Fropenem bound preferentially to both PBPs 2 and 1b and less strongly to PBP 1a. Fropenem exhibited low affinities for PBPs 3a and 3b, as did ritipenem. Cefdinir showed a high affinity for PBP 1b, for which the IC₅₀ was as low as that of ritipenem, and also showed high affinities for PBPs 3a and 3b, for which the IC₅₀s were 0.9 and 0.2 times the MIC, respectively. The affinity of cefdinir for PBP 2 was lower than those of ritipenem and fropenem. Cefsulodin has been well known as a selective inhibitor of PBP 1a in E. coli (13, 14), but cefsulodin was bound selectively to PBP 1b in the H. influenzae strain and exhibited a poor affinity for PBP 1a. Aztreonam and cefotaxime preferentially bound to PBPs 3a and 3b. Meropenem bound preferentially to PBP 2 and less strongly to PBPs 3b and 3a. Amoxicillin showed a high affinity for PBP 3b, followed by those for PBPs 1b, 2, and 3a. These results suggest that the primary inactivation of PBP 1b is essential to bacterial lysis in H. influenzae as well as in E. coli (12).

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Binding profiles of ritipenem and cefsulodin for PBPs of H. influenzae IID983. Membrane preparations of the strain were exposed to the indicated concentrations of each antibiotic. The estimated molecular mass (in kilodaltons) of each PBP is in parentheses.

Although ritipenem and cefsulodin, which led to drastic lysis, had low affinities for PBPs 3a and 3b, cefdinir, which has a lower lytic potency than ritipenem and cefsulodin, had moderate affinities for PBPs 3a and 3b. We performed an additional microscopic examination in order to investigate a possible involvement of binding to PBPs 3a and 3b in the lysis caused by ritipenem. The simultaneous addition of ritipenem and aztreonam, a PBP 3-selective inhibitor, to a bacterial culture at their MICs resulted in the formation of spindle-shaped cells; however, the cells evaded lysis. The evasion of lysis was also demonstrated in the culture treated with ritipenem and cefotaxime. These findings suggest that the inactivation of PBPs 3a and 3b may interfere with the bacterial lysis caused by the inactivation of PBP 1b in H. influenzae cells. This evidence differs from a previous report, which suggested that inactivation of PBPs 1a and 3 induced the lysis of H. influenzae cells (7). Fropenem, which has a lower lytic potency than ritipenem, also had low affinities for PBPs 3a and 3b, as did ritipenem, but had high affinities for both PBPs 2 and 1b. This finding suggests that the inactivation of PBP 2 may also interfere with lysis.

Our present investigation has led to the conclusion that the primary and predominant inactivation of PBP 1b without the inactivation of PBPs 3a and 3b is essential to rapid and drastic lysis of H. influenzae cells and that ritipenem possesses a characteristic profile of affinities for PBPs to induce rapid cell lysis.

The administration of some -lactams, such as ritipenem, cefsulodin, cefdinir, and fropenem, could lead to cell lysis, and rapid lysis can induce irreversible and rapid killing of bacteria without a release of highly inflammatory products as a result of cell wall degradation. Therefore, it has been suggested that -lactam antibiotics with strong lytic activity should be suitable for chemotherapy (14-16). Our present study may open up an important avenue for the further improvement of -lactams.