In vitro activities of piperacillin against beta-lactamase-negative ampicillin-resistant Haemophilus influenzae.

The in vitro activities of piperacillin (PIP) against beta-lactamase-negative ampicillin (AMP)-resistant (BLNAR) Haemophilus influenzae were compared with those of cefotaxime (CTX) and ceftriaxone (CRO), and the potency of PIP as therapy for meningitis caused by BLNAR is also discussed. PIP showed good activity (MIC at which 90% of strains are inhibited, 0.25 micro g/ml) against 69 BLNAR strains, and its activity was comparable to that of CRO and superior to that of CTX. No significant correlation was observed between the MICs of PIP and CTX or CRO or AMP, whereas a high correlation was observed between the MICs of CTX and CRO. In the killing study, PIP showed potent bactericidal activity compared with those of CTX and CRO. By microscopic examination, PIP caused the formation of a spindle and short filamentous cells with bulges and induced cell lysis in BLNAR strains, while treatment with CTX and CRO resulted in the formation of large, spherical cells without any obvious lysis. The affinity of Bocillin FL, a fluorescent penicillin used for determination of the 50% inhibitory concentration (IC(50)s) for penicillin-binding proteins (PBPs), to PBPs 3a and 3b of BLNAR strains was drastically decreased compared with that to an AMP-susceptible strain (ATCC 33391). In the case of the BLNAR strains, the IC(50)s for PBPs 1a, 1b, and 2 were similar to those for the PBPs of ATCC 33391. Since the affinity of binding to PBPs 3a and 3b of the BLNAR strains decreased drastically, the second targets among the PBPs were PBP 2 for PIP, PBP1 (1a and 1b) for CTX and CRO. In conclusion, PIP showed excellent activities against BLNAR strains in a manner different from those of cephem antibiotics, suggesting that it could be a candidate therapeutic agent for the treatment of meningitis caused by BLNAR strains.

In vitro and in vivo activities of T-705 and oseltamivir against influenza virus.

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has a potent and selective inhibitory activity against influenza virus. We studied the effects of an infectious dose on the anti-influenza virus activities of T-705 and oseltamivir, a commercially available neuraminidase inhibitor, both in vitro and in vivo. Plaque formation of influenza A/PR/8/34 virus was completely inhibited by 10 microg/ml of T-705 after 72 h incubation, whereas visible plaque formation was detected in the plate treated with GS 4071, the active form of oseltamivir (10 microg/ml). The antiviral activity of T-705 was not influenced by an increase in multiplicity of infection (MOI) from 0.0001 to 1, but that of GS 4071 was influenced in a yield reduction assay. No increase in viral yield was seen in either culture supernatant or cells after removal of T-705 (10 microg/ml) but, in contrast, productive infection recurred in culture supernatant and in cells after removal of GS 4071. In mice infected with a high challenge dose of influenza A/PR/8/34 virus, orally administered T-705 (200 and 400 mg/kg/day) completely prevented the death of mice and the survival rates of mice were significantly higher than those in mice treated with oseltamivir (P<0.01). When the treatment was delayed at 1, 13 and 25 h post infection, oral administration of 200 mg/kg of T-705 significantly prevented the death of mice (P<0.01), and the survival rates of mice treated with T-705 were comparable to those of mice treated with oseltamivir. These results suggest that T-705 has the potential to be a potent inhibitor of human influenza virus infections.

[Pharmacological properties and expected clinical role of an injectable new quinolone antibiotic, pazufloxacin mesilate].

Pazufloxacin mesilate (PZFX: Pasil INJECTION, Pazucross INJECTION) is a novel injectable quinolone antibiotic that was discovered by Toyama Chemical Co., Ltd. and codeveloped by Toyama Chemical Co. Ltd. and Mitsubishi Pharma Corporation. Laboratory studies indicated that PZFX reached high plasma levels quickly after intravenous administration, and it exhibited weak convulsion inducing activity, low local irritant effect, and less hypotensive activity, all of which are generally recognized as side effects associated with other injectable quinolones. PZFX has potent antibacterial and bactericidal activities against cephalosporin-, carbapenem-, and aminoglycoside-resistant strains. These favorable antibacterial and bactericidal activities gave PZFX superior therapeutic effects, compared to injectable cephalosporin antibiotics, on experimental animal infection models caused by those resistant strains. Clinical studies also revealed PZFX was as safe and effective as the injectable cephalosporin, ceftazidime, against various moderate to severe infections. Furthermore, PZFX showed good clinical effects on the infections resistant to chemotherapy by the other antimicrobial agents. These results indicate that PZFX is a viable choice for various bacterial infections. In this review, results of laboratory and clinical studies are summarized and the clinical role of PZFX among the injectable antimicrobials is discussed.

Evaluation of antimicrobial agents using an experimental pulmonary superinfection model with Aspergillus fumigatus and Pseudomonas aeruginosain leukopenic mice.

The therapeutic efficacy of amphotericin B (AmB), imipenem/cilastatin (IPM/CS), pazufloxacin (PZFX) mesilate, and combinations of these, was evaluated using an experimental pulmonary superinfection model in mice caused by Pseudomonas aeruginosa and Aspergillus fumigatus. The superinfected mice died within 3 days. Although the viable cell count of P. aeruginosa increased markedly from 10(3) to 10(8) CFU/lung on day 2 after infection, that of A. fumigatus decreased from 10(3) to 10(2) CFU/lung on that day, showing that P. aeruginosa facilitated the mortality in the superinfection. Extensive necrosis in the lung parenchyma and moderate hyphae proliferation of A. fumigatus were observed on day 2 after infection. Mice treated with PZFX mesilate (50 mg/kg per day) and the combination of PZFX mesilate (50 mg/kg per day) - AmB (2.5 mg/kg per day) showed prolonged survival in comparison to untreated control mice ( P < 0.05). In the PZFX mesilate-treated group, no significant necrosis was observed, but necrosis due to the hyphae proliferation of A. fumigatus was still observed in the lung parenchyma on day 6 after infection. However, neither significant necrosis nor hyphae proliferation of A. fumigatus was observed in mice treated with the combination of PZFX mesilate - AmB. On the other hand, the survival rates of mice treated with AmB (2.5 mg/kg per day), IPM/CS (50 mg/kg per day), and the IPM/CS-AmB combination were all less than 10%. The viable cell count of P. aeruginosa decreased in PZFX mesilate-alone group and in the combination of PZFX mesilate - AmB group, but no significant decrease in this count was observed in the IPM/CS and combination of IPM/CS-AmB group. The viable count of A. fumigatus was increased in the IPM/CS, PZFX mesilate-alone, and combination of IPM/CS-AmB groups, but the count was suppressed in the AmB-alone and the combination of PZFX mesilate - AmB group. In conclusion, this superinfection model would be useful to evaluate the therapeutic potential of combinations of antibacterial and antifungal agents, and the scheduling of drug administration in terminal infections caused by P. aeruginosa and A. fumigatus.

Mosaic-like structure of penicillin-binding protein 2 Gene (penA) in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime.

Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 micro g/ml) were isolated from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime and penicillin was transferred to a susceptible recipient, N. gonorrhoeae ATCC 19424, by transformation of the penicillin-binding protein 2 gene (penA) that had been amplified by PCR from a strain with reduced susceptibility to cefixime (MIC, 0.5 micro g/ml). The sequences of penA in the strains with reduced susceptibilities to cefixime were different from those of other susceptible isolates and did not correspond to the reported N. gonorrhoeae penA gene sequences. Some regions in the transpeptidase-encoding domain in this penA gene were similar to those in the penA genes of Neisseria perflava (N. sicca), Neisseria cinerea, Neisseria flavescens, and Neisseria meningitidis. These results showed that a mosaic-like structure in the penA gene conferred reductions in the levels of susceptibility of N. gonorrhoeae to cephems and penicillin in a manner similar to that found for N. meningitidis and Streptococcus pneumoniae.

[In vitro combination effect of pazufloxacin with various antibiotics against Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus].

The in vitro combination effects of pazufloxacin (PZFX) with various antibiotics were investigated by the checkerboard dilution method using piperacillin (PIPC), tazobactam/piperacillin (TAZ/PIPC), ceftazidime (CAZ), cefozoprane (CZOP), imipenem/cilastatin (IPM/CS), meropenem (MEPM), panipenem/betamipron (PAPM/BP), amikacin (AMK) and isepamicin (ISP) for clinical isolates of 27 Pseudomonas aeruginosa strains, vancomycin (VCM), teicoplanin (TEIC) and arbekacin (ABK) for clinical isolates of methicillin-resistant 26 Staphylococcus aureus (MRSA) strains, respectively. The following results were obtained. 1. For 27 P. aeruginosa strains, the synergistic effects were observed with the combination of PZFX and CAZ or MEPM (11.1%: 3 strains), and PZFX and CZOP or PAPM/BP (3.7%: 1 strain), respectively. The additive and synergistic effects of PZFX were observed with the combination in all beta-lactams tested in the strains more than 50%. No antagonistic effect was observed. The additive effects were also observed with the combination of PZFX and AMK or ISP in the strains more than 50% of the test strains and no antagonistic effect was observed. 2. For 26 MRSA strains, no antagonistic effect was observed with the combination of all antibiotics tested. The indifference was observed with the combination of PZFX and VCM or ABK in the strains more than 60%, and the additive effects were observed with the combination of TEIC in the strains more than 80%. In conclusion, no antagonistic effect was observed in PZFX with the combination of beta-lactams and anti-MRSA agents, suggesting that the combination therapy of PZFX with these antibiotics would be possible to use for the infections caused by P. aeruginosa and MRSA.

[In vitro and in vivo antibacterial activities of pazufloxacin mesilate, a new injectable quinolone].

We investigated the in vitro and in vivo antibacterial activities of pazufloxacin mesilate (PZFX mesilate), a new injectable quinolone, and obtained the following results. 1) The MIC50 and MIC90 values of PZFX against clinically isolated Gram-positive and -negative bacteria, ranged from 0.0125 to 12.5 micrograms/ml and 0.025 to 100 micrograms/ml, respectively. PZFX showed broad spectrum activity. The antibacterial activities of PZFX against quinolone-susceptible, methicillin-resistant Staphylococcus aureus, beta-lactamase-negative, ampicillin-resistant Haemophilus influenzae, extended spectrum beta-lactamase possessing Klebsiella pneumoniae and imipenem/cilastatine (IPM/CS)-resistant Pseudomonas aeruginosa were superior to those of ceftazidime (CAZ), ceftriaxone, IPM/CS, meropenem and panipenem/betamipron. 2) PZFX showed superior bactericidal activity against S. aureus, Escherichia coli, Proteus mirabilis, Serratia marcescens and P. aeruginosa to those of CAZ and IPM/CS after treatment for 15 minutes at the drug concentration equivalent to that in human serum at clinical dose to be continued for 15 minutes. 3) CAZ and IPM/CS had no bactericidal activity at the 16 times of MIC against P. aeruginosa in human polymorphonuclear leucocytes, while PZFX exhibited potent bactericidal activity in a dose-dependent manner against such bacteria. 4) PZFX inhibited both DNA gyrase and topoisomerase IV from S. aureus at nearly the same level. PZFX showed poor inhibitory activity against topoisomerase II from human placenta and showed high selectivity to bacterial topoisomerase. 5) PZFX mesilate showed superior therapeutic activity to that of CAZ with following infection model caused by S. aureus and P. aeruginosa or each; systemic infection with cyclophosphamide-treated mice, systemic infection in mice with high challenge doses, CMC pouch infection in rat, and calculus infection in rat bladder. 6) Intravenous administration of PZFX with high plasma concentration just after administration, showed more excellent therapeutic effect against the rat intraperitoneal infection, than p.o. and s.c. administration.

Evaluation of T-3811ME (BMS-284756), a new des-F(6)-quinolone, for treatment of meningitis caused by penicillin-resistant Streptococcus pneumoniae in rabbits.

T-3811ME (BMS-284756) is a new des-F(6)-quinolone with high levels of activity against gram-positive bacteria, including penicillin-resistant Streptococcus pneumoniae (PRSP) strains. T-3811, the free base of T-3811ME, exhibited potent activity against 28 clinical strains of PRSP isolated clinically (MIC at which 90% of the isolates tested are inhibited, 0.0625 microg/ml). After the intravenous dosing of T-3811ME (20 mg/kg of body weight as T-3811) in rabbits with meningitis caused by PRSP, the area under the concentration-time curve (AUC) of T-3811 in cerebrospinal fluid (CSF) was 5.79 microg. h/ml and was 4.5-fold higher than that of T-3811in the CSF of rabbits without meningitis. In addition, the AUC/MIC for T-3811ME (20 mg/kg as T-3811) in CSF was 185, which was 4.3-fold higher than that for ceftriaxone (administered intravenously at 100 mg/kg). After the administration of any dose of T-3811ME (5, 10, and 20 mg/kg as T-3811), the viable cell counts in CSF decreased in a dose-dependent manner. In particular, after dosing of 20 mg/kg (as T-3811), the viable cell counts in CSF were significantly less than those in the nontreated group (P < 0.01). By histopathological evaluation, 6 h after the administration of T-3811ME (20 mg/kg as T-3811), the thickening of the cerebral meninx and the infiltration of neutrophils into the cerebral meninx were less severe in the treated group than in the nontreated group. T-3811ME (BMS-284756) may be expected to be evaluated for the management of meningitis caused by highly penicillin-resistant pneumococci.

[In vitro interaction of tazobactam/piperacillin combined with aminoglycosides against Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli AND Staphylococcus aureus].

The in vitro combination effect of tazobactam/piperacillin (TAZ/PIPC) with aminoglycosides (amikacin (AMK) and isepamicin (ISP)) were investigated by the checkerboard dilution method against PIPC-resistant and TAZ/PIPC-susceptible Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Methicillin-sensitive Staphylococcus aureus (MSSA). The following results were obtained. 1. The combination of TAZ/PIPC with AMK showed synergistic effect for 66.7% of P. aeruginosa and 9.1% of K. pneumoniae and additive effect for 76.9% of E. coli and 74.1% of MSSA. The antagonistic effect of TAZ/PIPC with AMK was not demonstrated for all tested strains. 2. The combination of TAZ/PIPC with ISP showed synergistic effect for 61.9% of P. aeruginosa and 22.7% of K. pneumoniae and additive effect for 84.6% of E. coli and 66.7% of MSSA. The antagonistic effect of TAZ/PIPC with ISP was not demonstrated for all tested strains. In conclusion, these results suggest that the combination therapies of TAZ/PIPC with aminoglycosides are useful for the clinical treatment of sepsis caused by above four species.

Muscle distribution of antimicrobial agents after a single intravenous administration to rats.

The purpose of this study was to evaluate the distribution of three fluoroquinolones (pazufloxacin, ciprofloxacin and ofloxacin) and a beta-lactam, ceftazidime in the tissue interstitial and intracellular spaces after a single intravenous administration to rats based on muscle microdialysis. The unbound concentration in the tissue interstitial fluid (C(isf,u)) after administration was estimated from the concentration in the dialysate by muscle microdialysis, the in vitro permeability rate constant, and the previously reported effective dialysis coefficient. The C(isf,u)s of pazufloxacin, ciprofloxacin, ofloxacin and ceftazidime in the muscle were close to their unbound concentrations in the venous plasma. These results were consistent with ones previously obtained at steady state. Based on these results, the total concentration in the tissue interstitial fluid (C(isf)) was calculated from the ratio of plasma protein binding, the plasma concentration, and previously reported interstitial-to-plasma albumin ratio in muscle of rats. The calculated C(isf) was compared with the muscle concentration (C(m)) obtained using the homogenized tissue. The C(isf) of ceftazidime was higher than the C(m). The C(isf) of pazufloxacin was found to be almost equal to its C(m). The C(isf)s of ciprofloxacin and ofloxacin were lower than their C(m)s with the exception of the values at 5 min after administration. These results indicate that ceftazidime is mainly distributed in the interstitial space of the muscle, that pazufloxacin is distributed equally in both the interstitial space and the tissue cells, and that ciprofloxacin and ofloxacin are mainly distributed in the tissue cells rather than the interstitial space.

A plasmid-mediated beta-lactamase with carbapenem-hydrolyzing activity from Serratia marcescens.

Serratia marcescens W-313/pSW313 was isolated from a clinical specimen that showed resistance to various beta-lactams. beta-Lactamase from this strain hydrolyzed oxyiminocephalosporins and carbapenems, but did not hydrolyze 2-carboxypenam and aztreonam. The kinetic parameters of this enzyme were similar to those of the carbapenem-hydrolyzing beta-lactamases mediated by R-plasmid from Pseudomonas aeruginosa and S. marcescens, whose properties were previously reported.