Synergistic effects of a macrolide and a cell wall-affecting antibiotic on Pseudomonas aeruginosa in vitro and in vivo. 2. Combined effects of a macrolide with a fosfomycin and an aminoglycoside antibiotic

Synergistic effects of the cell wall-affecting antibiotics, dibekacin (DKB) and fosfomycin (FOM) and a macrolide antibiotic, midecamycin (MDM) or its derivative 9,3"-di-O-acetylmidecamycin (MOM) against Pseudomonas aeruginosa were investigated in vitro and in vivo. Synergistic effects were evaluated by estimating the number of viable bacteria at varying intervals after the two kinds of antibiotics were added to the logarithmic phase of the bacterial solution. Six hours after addition of antibiotic, the viable bacterial count of the culture treated with FOM and MOM underwent 2 log reduction compared to that which treated with FOM alone. Thus synergistic effect was significant. The number of viable bacteria treated with DKB and MDM showed slight reduction at 3 hours after addition of the two antibiotics and a marked reduction was noted after 20 hours compared with the control. Synergistic action was also demonstrated in in vivo experiments using mice. Three experimental mouse infection models, intraperitoneal infection, subcutaneous infection with carrageenan solution and burn infection were used. FOM was administered subcutaneously. DKB was administered intramuscularly. MDM or MOM was administered by the oral route. In all three experiments the survival rate of infected mice treated with FOM and MOM increased significantly compared to control mice. Similar synergistic effect was also obtained with DKB and MDM.     

Synergistic effects of a macrolide and a cell wall-affecting antibiotic on Pseudomonas aeruginosa in vitro and in vivo. 3. Incorporation of [14C]midecamycin acetate (MOM) into P. aeruginosa pretreated with cell wall-affecting antibiotics

The occurrence in beta-lactam treated patients of unstable L-forms of Pseudomonas aeruginosa insensitive to various antibiotics and synergistic effect of combined action of cell wall-affecting antibiotics and macrolide on Pseudomonas infection led us to examine the effects of macrolide on P. aeruginosa pretreated with cell wall-affecting antibiotics. The effects of macrolide antibiotics such as midecamycin acetate (MOM) on P. aeruginosa was investigated, a rapid killing effect by MOM was noted after treatment with suboptimal doses of cell wall-affecting antibiotics such as polymyxin B, carbenicillin, dibekacin or fosfomycin. Incorporation of [14C]MOM into intact P. aeruginosa cells was not significant, but was apparent into L-form cells or cells pretreated with cell wall-affecting antibiotics. The incorporated radioactivity was found in the 70 S ribosome fraction, binding with the 50 S subunits of ribosome in both cases. These results indicate that under certain conditions a macrolide antibiotic can enter the P. aeruginosa cell.     

Toxicological studies on a new macrolide antibiotic, midecamycin acetate (miokamycin). Part III-2. Chronic toxicity in beagle dogs: 13-week-treatment in male dogs

Miokamycin (MOM) is a derivative of midecamycin, a macrolide antibiotic isolated from a culture broth of Streptomyces mycarofaciens. The objective of this study was to determine the chronic toxicity of MOM in male Beagle dogs after once daily repeated p.o. administration of MOM, non-crystalline solid, for 13 weeks at daily dosage of 200 and 400 mg/kg. It is concluded that no toxic effects of MOM, non-crystalline solid, were shown in the present studies. Therefore, the maximum non-toxic dose is presumed to be 400 mg/kg or more in male Beagle dogs administered p.o. once daily for 13 weeks.     

Mode of action of the protein, SP127, which enhances the activity of macrolide antibiotics against Pseudomonas aeruginosa.

Antibiotics, the activity of which enhanced against Pseudomonas aeruginosa by SP127, were restricted to the basic macrolide antibiotics such as erythromycin, maridomycin and oleandomycin, the neutral macrolide antibiotics such as lankamycin and lankacidin C, vancomycin and enramycin. Synergistic activity of SP127 with the above antibiotics was found against Pseudomonas aeruginosa and several strains of Escherichia coli, but not against Proteus vulgaris and macrolide-resistant Staphylococcus aureus. SP127 had extremely weak proteolytic but no lytic activity. From the isotopic experiments, the action of SP127 was partially attributed to the promotion of antibiotic penetration to cells of Pseudomonas aeruginosa.     

Toxicological studies on a new macrolide antibiotic, midecamycin acetate (miokamycin). Part III-3. Chronic toxicity in beagle dogs: 26-week-treatment in female dogs

Miokamycin (MOM) is a derivative of midecamycin, a macrolide antibiotic isolated from a culture broth of Streptomyces mycarofaciens. The objective of this study was to determine the chronic toxicity of MOM in female Beagle dogs after once daily repeated p.o. administration of MOM, non-crystalline solid, for 26 weeks at daily dosages of 100, 200 and 400 mg/kg. It is concluded that MOM, non-crystalline solid, did not show any toxic effects in this study and the maximum non-toxic dose is presumed to be 400 mg/kg or more in female Beagle dogs administered once daily for 26 weeks.     

Synergistic activity of isepamicin and beta-lactam antibiotics against Pseudomonas aeruginosa in vitro and in vivo

Synergistic activities of isepamicin (ISP) and a beta-lactam antibiotic such as piperacillin (PIPC) or cefotaxime (CTX) against Pseudomonas aeruginosa were demonstrated in vitro and in vivo. In vitro synergistic activity was observed when ISP was used together PIPC or CTX. The synergy observed in vitro was reproduced in vivo against experimental mouse infections, and a ISP-PIPC or a ISP-CTX combination showed significantly greater protective effects than individual antibiotics by themselves.     

Toxicological studies on a new macrolide antibiotic, midecamycin acetate (miokamycin). Part II-1. Subacute toxicity in rats

Miokamycin (MOM) is a derivative of midecamycin, a macrolide antibiotic isolated from a culture broth of Streptomyces mycarofaciens. The objective of this study was to determine the subacute toxicity in male and female rats (Wistar, SPF, 5-week-old) after repeated oral administration of MOM, non-crystalline solid, for 5 weeks at selected dosage levels of 1,000, 2,000 and 4,000 mg/kg/day. It is concluded that the maximum non-toxic dosage level of MOM, non-crystalline solid, was 1,000 mg/kg/day but without specific toxic effects with rats when it was orally administered once daily for 5 weeks.     

Clinical studies on 9, 3"-diacetylmidecamycin in respiratory tract infections in the field of pediatrics (author's transl)

Laboratory and clinical studies were performed on 9, 3"-diacetylmidecamycin (MOM), a new macrolide antibiotic in the field of pediatrics, and the results were as follows. Antibacterial activity: For 32 clinically isolated strains of Staphylococcus aureus, the MIC of MOM ranged from 0.78 to 1.56 micrograms/ml for 17 of the 32 strains, and exceeded 100 micrograms/ml for the 15 remaining strains with both inoculum sizes of 10(8) cells/ml and 10(6) cells/ml. For 27 strains of Streptococcus pyogenes, the MIC range was wide, varying from 0.10 to greater than or equal to 100 micrograms/ml and less than 1.56 micrograms/ml for about 2/3 of all the 27 strains. For 9 strains of Bordetella pertussis, the MIC ranged from 0.10 to 0.78 microgram/ml and 0.10 to 0.39 microgram/ml with the inoculum size of 10(8) cells/ml and 10(6) cells/ml, respectively. Comparing the antibacterial activity of MOM with that of midecamycin (MDM) and erythromycin (EM) against these 3 bacterial species, MOM was almost comparable to MDM, but about 2 or 3 tubes inferior to EM. Absorption and excretion: MOM was administered to 5 children (from 5 to 8 years old) at a dose of 10 mg/kg or 20 mg/kg at 30 minutes before breakfast. The peak of serum concentration was observed 30 minutes to 1 hour after administrations of both dosages: 0.52 to 1.71 micrograms/ml with 10 mg/kg and 0.88 to 1.77 micrograms/ml with 20 mg/kg. 0.09 to 1.10% and 0.94 to 1.19% of MOM were excreted in the urine within the first 6 hours, respectively. Clinical results: MOM was administered to 28 pediatric patients with acute respiratory tract infections (acute pharyngitis; 2, acute purulent tonsillitis; 19, acute bronchitis; 4, acute pneumonia; 2 and whooping cough; 1). The overall clinical response was excellent in 10, good in 10, fair in 3 and poor in 5; the efficacy rate was 71.4%. Isolated S. pyogenes strains were eradicated in 6 out of 11 strains, reduced in 3 and unchanged in 2 strains. One strain of S. aureus was eradicated. One strain of non group A beta-Streptococcus was reduced. Haemophilus influenzae strains were reduced in 1 of the 4 strains and unchanged in 3 strains. The overall eradication rate was 41.2%. No side effects or abnormal laboratory findings were observed, but 1 case complained of a bitter taste.     

The combined effects of aspoxicillin with aminoglycoside antibiotics on Pseudomonas aeruginosa

Combined actions of aspoxicillin (ASPC) with several aminoglycosides (AGs) against various Pseudomonas aeruginosa strains were examined using the checker board method and experimental infection of mice, and the actions were compared with those of piperacillin (PIPC) and mezlocillin (MZPC). 1. The combination of ASPC with gentamicin, amikacin (AMK) or tobramycin showed synergistic activities against 81.9-95.5% of the test strains. These frequencies were higher than those of reference penicillins (PCs). Mean values of FIC index for combinations between ASPC and AGs were smaller than 0.5, thus, the combinations showed the strongest synergism among the PCs tested. 2. ASPC combined with AGs showed synergistic actions on experimental mouse infections caused by strains of P. aeruginosa. The potency of ASPC was the same as that of PIPC, but MZPC had a weaker activity than ASPC or PIPC. 3. Schedule of administration of ASPC and AMK was examined using experimental infection in mice caused by P. aeruginosa. When AMK was administered first, a synergism was clearly observed when ASPC was administered within 1 hour of the AMK administration. When ASPC was administered first, a synergism was observed when AMK was administered within 4 hours of the ASPC administration. 4. Influences of AMK and ASPC or reference PCs on growth of P. aeruginosa 22 were examined at lower concentrations than MIC. AMK showed a bacteriostatic action on the test strain at 1/4 MIC. But no influence was observed at lower concentrations than 1/4 MIC of AMK. ASPC and reference PCs showed slight effects on growth of the test strain at concentrations of 1/32 MIC of PIPC, 1/128 MIC of MZPC and 1/256 MIC of ASPC. The PCs showed bactericidal action against the test strain at these concentrations when combined with 1/4 MIC of AMK.     

Toxicological studies on a new macrolide antibiotic, midecamycin acetate (miokamycin). Part III-1. Chronic toxicity in rats

Miokamycin (MOM) is a derivative of midecamycin, a macrolide antibiotic isolated from a culture broth of Streptomyces mycarofaciens. The objective of this study was to determine the chronic toxicity of MOM in male and female rats (Wistar, SPF, 5-week-old) after repeated oral administration of MOM, non-crystalline solid, for 26 weeks at daily dosages of 62.5, 125, 250, 500 and 1,000 mg/kg. The lowest dosage level of 62.5 mg/kg/day was only applied for female rats. In conclusion, the maximum non-toxic dosage level of MOM, non-crystalline solid, is presumed to be 250 mg/kg in male and female rats with p.o. administered once daily for 26 weeks.     

Combination effects between KW-2228 and antibiotics against systemic infections in normal and neutropenic mice]

KW-2228, a mutationally modified recombinant human granulocyte colony-stimulating factor (rhG-CSF), possesses some excellent properties such as high specific activity in stimulating granulocyte colony-formation in vitro, great biological stability in plasma, good pharmacokinetic profile and high potency in granulopoiesis in normal mice in vivo. Recently, the application of G-CSF against infectious diseases has been considered, and some animal experiments have been carried out to support such an application on human infectious diseases. In this paper, we examined combination effect of KW-2228 with various chemotherapeutic drugs in experimental infectious in mice. A combination effect of KW-2228 with ceftazidime (CAZ) was evaluated in a systemic infection with Pseudomonas aeruginosa in normal mice. Combination effects of KW-2228 with CAZ, astromicin and amphotericin B were also evaluated in experimental systemic infections caused by P. aeruginosa, Serratia marcescens and Candida albicans in immunosuppressed mice treated with cyclophosphamide. Synergistic effects were generally observed at KW-2228 doses from 1 to 5 micrograms per mouse with all combinations. We concluded that combination therapies of KW-2228 with various chemotherapeutic drugs in experimental infections in mice showed that it should be effective in normal and immunosuppressed host. These results of our laboratory studies suggest that KW-2228 in combination with antibiotics would be useful in the clinical treatment of microbial infections. Recently, clinical efficacy studies of KW-2228 have been initiated in Japan.     

In vivo antibacterial activity of cefodizime, a new cephalosporin antibiotic

The in vivo activity of cefodizime (HR 221) was compared with that of cefotaxime (CTX), cefmenoxime, latamoxef, cefazolin and cefmetazole (CMZ). The protective effects of HR 221 on experimental infections in mice caused by Staphylococcus aureus Smith, Escherichia coli C-11, Proteus vulgaris GN-76 and Serratia marcescens No. 2 were directly related to its in vitro activity against these strains. In contrast, the compound showed the smallest ED50 values, among the 5 antibiotics tested (not including CMZ), for Klebsiella pneumoniae 3K-25 and Pseudomonas aeruginosa PI 67 against which it had relatively low in vitro activity, and its ED50 for Citrobacter freundii GN-346 was as small as 1.821 mg/mouse in spite of its MIC of greater than 100 micrograms/ml. HR 221 exerted potent bactericidal activity against Streptococcus pneumoniae Sp-1 inoculated into the mouse lung; the duration of action was prolonged. When tested against the E. coli Ec-89 infection induced in the rat uterus, the activity of HR 221 given to rats once daily was equal to that of CTX or CMZ given at the same dose twice daily.     

Ultrastructure of Mycoplasma pneumoniae exposed to macrolide antibiotics during cultivation on a glass surface and in an organ culture, in relation to their mycoplasma-killing activity

The macrolide antibiotics midecamycin acetate (MOM), erythromycin (EM), midecamycin (MDM), josamycin (JM) and rokitamycin (RKM) showed killing activity against Mycoplasma pneumoniae strain FH-P24. The activity of MOM, EM and JM was not influenced by the number of organisms inoculated, but that of RKM was markedly decreased by a large inoculum. Scanning electron microscopic observations showed many long filaments crossing over each other with small colonies when the organisms were cultivated on a glass surface without any drug for 72 h. When they were exposed to four times the MIC of MOM, the filamentous forms were decreased and the colonies did not grow to a large size. However, after exposure to the other macrolides, the colonies grew larger. Transmission electron micrographs revealed that intracellular vacuolization of the organisms was induced by exposure to MOM, EM and JM, but a mixture of vacuolized cells and "young" organisms was observed after exposure to MDM and RKM. In hamster tracheal organ cultures, the number of organisms was greatly decreased by exposure to four times the MIC of MOM, and the remaining filamentous and colonized organisms were lysed. However, treatment with four times the MIC of the other macrolides induced hardly any lysis of the organisms. In transmission electron micrographs, filamentous and rounded organisms filling the ribosome-like matrix could be seen on the epithelial surface. Treatment with four times the MIC of these macrolides decreased the number of organisms and vacuolized filamentous organisms could be seen on the epithelial cells.     

Toxicological studies on a new macrolide antibiotic, midecamycin acetate (miokamycin). Part I-1. Acute toxicity in mice

Miokamycin (MOM) is a derivative of midecamycin, a macrolide antibiotic isolated from a culture broth of Streptomyces mycarofaciens. The objective of this study was to determine the acute toxicity of MOM, non-crystalline solid, in male and female mice (Jcl-ICR, SPF, 5-week-old) after single i.p., and p.o. administration of this material at a dose level of 5,000 mg/kg as the maximum physically applicable dose. MOM, non-crystalline solid, exhibited no acute toxicity in the present study. LD0 values were estimated as more than 5,000 mg/kg in each route of administration.     

In vitro combination effects of astromicin and beta-lactam antibiotics against CFS-sensitive and CFS-resistant Pseudomonas aeruginosa. In vitro synergistic activity

We investigated in vitro synergistic activity of astromicin (ASTM) combined with beta-lactam antibiotics (cefsulodin (CFS), cefoperazone (CPZ), ceftazidime (CAZ), piperacillin (PIPC) and fosfomycin (FOM) against fresh clinical isolated Pseudomonas aeruginosa, which consisted of 13 CFS sensitive (MIC less than or equal to 3.13 micrograms/ml) and 19 CFS resistant (MIC greater than or equal to 400 micrograms/ml) strains according to the FIC index. Against CFS-sensitive P. aeruginosa, ASTM showed good synergistic activities when combined with PIPC (54%), CAZ (38%), CPZ (23%) and CFS (8%). Against CFS-resistant P. aeruginosa, ASTM also showed high synergistic activities when combined with CAZ (63%), CPZ (47%), PIPC (37%) and CFS (11%). Among the CFS-resistant P. aeruginosa, one clinical isolate showed a high sensitivity (MIC0.78 micrograms/ml) against ASTM alone.     

Combination antibacterial effects between aztreonam and eight other antibiotics

In vitro interactions between aztreonam (AZT) and 8 other antibiotics were studied using the agar dilution checkerboard technique against 88 clinical isolates of Escherichia coli, Proteus vulgaris, Serratia marcescens and Pseudomonas aeruginosa. Combinations of AZT with 8 other antibiotics were generally additive or indifferent. Synergism was occasionally seen against S. marcescens or P. aeruginosa with AZT plus isepamicin (ISP). Antagonism was observed only between AZT and latamoxef against P. vulgaris. In a phase-contrast microscopic study, synergistic effects between AZT and aspoxicillin or ISP were confirmed against E. coli 177 and P. aeruginosa 15846. AZT in combination with ISP demonstrated a synergy against experimental urinary tract infection in mice caused by P. aeruginosa 15846. We believe that combinations of several antibiotics with AZT should be considered for initial therapy of infections because synergism and additive action were observed and antagonism was rarely found in our study.     

Synergistic activity of astromicin and beta-lactam antibiotics against Pseudomonas aeruginosa in vitro and in vivo

Synergistic activity of astromicin and an antipseudomonal beta-lactam antibiotic such as piperacillin, cefsulodin or carbenicillin against Pseudomonas aeruginosa was demonstrated in vitro and in vivo. Synergy in vitro was observed more often when astromicin was combined with piperacillin or cefsulodin than when it was combined with carbenicillin. The combination of astromicin with piperacillin showed a bactericidal activity against Pseudomonas aeruginosa at a bacteriostatic concentration of each antibiotic alone. The synergy observed in vitro was reproduced against experimental mouse infections, and the astromicin-piperacillin or cefsulodin combination produced significantly greater protective effects than the single use of individual antibiotics.     

In vivo combination effects of astromicin and beta-lactam antibiotics against Pseudomonas aeruginosa

Astromicin (ASTM, Fortimicin) is a pseudodisaccharide aminoglycoside antibiotic. The ASTM exhibited excellent activity against Gram-positive and Gram-negative bacteria but was only weakly active against Pseudomonas aeruginosa. In vitro synergistical activities of ASTM combined with beta-lactam antibiotics have been reported against P. aeruginosa previously. In this paper, we investigated the in vivo combination efficacies of ASTM and beta-lactam antibiotics (latamoxef (LMOX), cefoperazone (CPZ), piperacillin (PIPC) and cefsulodin (CFS) against experimental infection with P. aeruginosa in both normal and immunosuppressed mice. In normal mice, the combination of ASTM with these beta-lactam antibiotics produced significantly greater protective effects than the single use of individual antibiotics against both strains of P. aeruginosa BMH No. 1 and E-2. In mice immunosuppressed with cyclophosphamide, the combination of ASTM with LMOX or CFS also exhibited synergistic protective effects against P. aeruginosa BMH No. 1, but PIPC and CPZ did not. From the above results, the combination therapy of ASTM with beta-lactam antibiotics appeared to be effective against experimental infections with P. aeruginosa in mice.     

The comparison of in the vitro effect of imipenem or meropenem combined with ciprofloxacin or levofloxacin against multidrug-resistant Pseudomonas aeruginosa strains

This study evaluated the in vitro effects of the combination of a carbapenem (imipenem or meropenem) with a quinolone (ciprofloxacin or levofloxacin) using a microbroth dilution chequerboard technique and multidrug-resistant Pseudomonas aeruginosa strains. The ciprofloxacin and meropenem combination was only synergistic against 2 strains (6.2%) and ciprofloxacin and imipenem against 1 strain (3.1%). Levofloxacin and imipenem or meropenem were not synergistic for against any strain. None of the combinations showed an antagonistic effect.     

The combined activity of ampicillin with streptomycin or chloramphenicol against Pseudomonas aeruginosa

The bacteriostatic and bactericidal activity of ampicillin when combined at ten different ratios with either streptomycin or chloramphenicol against Pseudomonas aeruginosa has been investigated. Synergistic bacteriostatic effect was obtained with all ampicillin/streptomycin combinations and 9 ratios of ampicillin/chloramphenicol combinations. The highest synergistic value was obtained by the ratios containing 98% of ampicillin and 2% of either streptomycin or chloramphenicol. A synergistic and an indifferent combined bactericidal effect was obtained with ampicillin/streptomycin and ampicillin/chloramphenicol combinations respectively. The mechanism of such synergism is briefly discussed.