Antimicrobial peptides contribute to innate host defense against a number of bacteria and fungal pathogens. Some of antimicrobial synthetic peptides were systemically administered in vivo; however, effective protection has so far not been obtained because the effective dose of peptides in vivo seems to be very high, often close to the toxic level against the host. Alternatively, peptides administered in vivo may be degraded by certain proteases present in serum. In this study, D‐amino acids were substituted for the L‐amino acids of antimicrobial peptides to circumvent these problems. Initially a peptide (L‐peptide) rich in five arginine residues and consisting of an 11‐amino acid peptide (residues 32–42) of human granulysin was synthesized. Subsequently, the L‐amino acids of the 11‐amino acid peptide were replaced partially (D‐peptide) or wholly (AD‐peptide) with D‐amino acids. Activity and stability to proteolysis, in particular, in the serum of antimicrobial peptides with D‐amino acid substitutions were examined. Peptides with D‐amino acid substitutions were found to lyse bacteria as efficiently as their all‐L‐amino acid parent, L‐peptide. In addition, the peptide composed of L‐amino acids was susceptible to trypsin, whereas peptides containing D‐amino acid substitutions were highly stable to trypsin treatment. Similarly, the peptide consisting of L‐amino acids alone was also susceptible to fetal calf serum (FCS), however, protease inhibitors restored the lowered antimicrobial activity of the FCS‐incubated peptide. Thus, D‐amino acid substitutions can make antimicrobial peptides resistant to proteolysis, suggesting that the antimicrobial peptides consisting of D‐amino acids are potential candidates for clinical therapeutic use.

A total of 105 isolates of Mycoplasma pneumoniae were evaluated for susceptibility to moxifloxacin, sparfloxacin, levofloxacin, and ciprofloxacin. Moxifloxacin, a newly synthesized compound, showed the greatest activity. The MICs and MBCs at which 50 and 90% of isolates were affected were 0.15 (MIC 50 and MBC 50 ) and 0.3 g/ml (MIC 90 and MBC 90 ) respectively. The results indicate that moxifloxacin might be promising an antimycoplasmal agent.Mycoplasma pneumoniae is the causative agent of upper respiratory tract infections and pneumonia in humans (6). Recently the efficacy of new quinolones has been demonstrated in vitro against various respiratory pathogens, including M. pneumoniae (2,4,11). In this report, we compared the in vitro activity of moxifloxacin, a new member of the fluoroquinolone group which has potent antimicrobial activities against grampositive and gram-negative bacteria (3, 9), with those of other fluoroquinolone as references against M. pneumoniae.A total of 105 strains of M. pneumoniae isolated from throat swabs from patients with pneumonia from 1986 (and earlier) to 2000 were used. These strains were subcultured fewer than five times in liquid medium and stored at Ϫ80°C until use. The antimicrobial agents used were moxifloxacin (Bay12-8039; Bayer Yakuhin Ltd.), sparfloxacin (SPFX; Dainippon Pharmaceutical Co., Ltd.) levofloxacin (LVFX; Daiichi Pharmaceutical Co., Ltd.), and ciprofloxacin (CPFX; Bayer Yakuhin Ltd.). The MICs and MBCs against each drug were determined by serial dilutions of the drugs in broth medium as described previously (5,8). Briefly, each strain of M. pneumoniae cultured for 5 days was diluted to 10 5 CFU/ml and 200 l of each sample was cultured in a sealed microtiter plate (Nunc Co., Ltd., Roskilde, Denmark) at 37°C. After 2 days of cultivation, twofold dilutions of the antimicrobial agents were added to samples of each strain. After an additional 2 days of cultivation, two and three 10-fold dilutions of each sample were used for plating on agar plates to provide a range of 500 to 1,000 CFU/10 l of the sample. The number of mycoplasmas in broth without drugs reached approximately 10 7 CFU/ml. MIC is defined as the lowest concentration of an antimcrobial agent inhibiting more than 99% of the mycoplasmal colonies with the control. The definition of MBC most often used in clinical microbiology is the lowest drug concentration that kills 99.9% of the bacterial population in a liquid medium.As shown in Fig. 1a, moxifloxacin (MIC for 50% of isolates [MIC 50 ], 0.15 g/ml; MIC for 90% of isolates [MIC 90 ], 0.3 g/ml) was more active than SPFX (MIC 50 , 0.3 g/ml; MIC 90 , 1.25 g/ml), LVFX (MIC 50 , 1.25 g/ml; MIC 90 , 2.5 g/ml), and CPFX (MIC 50 , 5.0 g/ml, MIC 90 , 5.0 g/ml). The MBCs and their distributions among the strains for these agents are shown in Fig. 1b; the MBCs of moxifloxacin (MBC 50 , 0.15 g/ml; MBC 90 , 0.3 g/ml) were markedly lower than those of SPFX (MBC 50 , 0.6 g/ml; MBC 90 , 2.5 g/ml), LVFX (MBC 50 , 2.5 g/ml; MBC 90 , 5.0 g/ml), and CPFX (MBC 50 , 5.0 g/m...

Cationic peptides are known to play critical roles in innate immunity. The peptides exert not only antimicrobial activity but also suppress the activity of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) by binding to them. We have previously reported that L-peptide, a small linear cationic peptide derived from human granulysin displays broad-spectrum antimicrobial activity. In this study, the in vitro interactions of L-peptide with LPS and LTA were examined. LPS and LTA were found to inhibit the antimicrobial activity of the L-peptide in a dose-dependent manner, and they were shown to bind with the L-peptide. On the other hand, L-peptide failed to inhibit LPS-or LTA-induced cytokine production by macrophages or to block the binding of LPS to the cell surface. Thus, there seems to be a hierarchy that places LPS and LTA above L-peptide in the interactions of L-peptide with LPS and LTA.

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