Synergistic effects of a macrolide and a cell wall-affecting antibiotic on Pseudomonas aeruginosa in vitro and in vivo. 1. Combined effects of a macrolide and a peptide antibiotic

Synergistic effects of peptide and macrolide antibiotics against Pseudomonas aeruginosa were investigated in vitro and in vivo. Synergistic effects were evaluated by estimating the number of viable bacteria at varying intervals in the logarithmic growth phase. These bacteria were treated concurrently with polymyxin B (PL) at the final concentration of 1.56 U/ml and with 9,3"-di-O-acetylmidecamycin (MOM) at varying concentrations. Synergistic effect was observed when PL was used with MOM at 3.13 and 12.5 microgram/ml respectively. When MOM at 50 microgram/ml was used with PL, the viable bacterial count was reduced to below 1/300 of the control to which PL alone had been added. Thus, the synergistic effect was remarkable. Similar results were obtained when colistin methanesulfonate (CL) was used instead of PL. Subsequently, attempts were made to determine if this action could also be found in in vivo experiments using mice. PL or CL was injected intramuscularly and midecamycin (MDM) or MOM was administered once or repeatedly by the oral route. Simultaneously, Pseudomonas aeruginosa strain IFO 3455 was inoculated intraperitoneally to mice. In the case of treatment once or repeatedly using both PL and MDM or MOM, the survival rate of infected mice increased significantly compared to single treatment by PL alone. Thus, the synergistic effects were demonstrated in four experiments. (The significance levels for the experiments were P = 0.070, 0.015, 0.042 and 0.024). Similar results were obtained when strain No. 5 was used to infect mice (P = 0.0096, 0.0027). When CL and MOM were given to mice once prior to infection with strain No. 5, synergistic effects were obtained as well (P = 0.010, 0.034).     

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.     

In vitro combination effects of doripenem with aminoglycoside or ciprofloxacin against Pseudomonas aeruginosa

This study evaluated the in vitro activity of combinations of doripenem (DRPM) with aminoglycosides (tobramycin or amikacin) or fluoroquinolone (ciprofloxacin) against 92 isolates of Pseudomonas aeruginosa from 16 clinical facilities in 2004 in Japan. We also tested combination effect of other carbapenems (imipenem (IPM), meropenem, biapenem) with aminoglycosides or fluoroquinolone by checkerboard dilution methods. DRPM showed synergistic or additive effects with the aminoglycosides or the fluoroquinolone against 90% of the isolates. The combination of DRPM and aminoglycosides showed the strongest synergistic effects against IPM-intermediate resistant and IPM resistant strains among the tested combinations. These results suggested that combination of DRPM with aminoglycosides would be useful for the treatment of infections caused by P aeruginosa including IPM-resistant strains.     

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.     

Combined effects of arbekacin with biapenem against in vitro and in vivo model of a mixture of MRSA and Pseudomonas aeruginosa

Combined effects of arbekacin (ABK) with biapenem (BIPM) were examined on both in vitro and in vivo model of a mixture of MRSA and Pseudomonas aeruginosa. As a result, significant effect in vitro was observed in combined use of ABK (1/2 MIC) with BIPM (1/4 and 1/2 MIC) against MRSA as compared with ABK or BIPM alone. Against P. aeruginosa combined effect was also observed, showing reduction of viable cells to the limitation of detection within 2 hours. Moreover, with respect to the protective effect on mixed systemic infection of MRSA and P. aeruginosa, the combined treatment with ABK and BIPM showed more excellent efficacy as compared with the single use of each drug.     

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.     

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.     

In vitro investigations on the action fosfomycin alone and in combination with other antibiotics on Pseudomonas aeruginosa and Serratia marcescens

86.4% of the Pseudomonas aeruginosa and 91% of the Serratia marcescens strains were sensitive to fosfomycin. In combination with mezlocillin, cefoxitin, gentamicin and nalidixic acid a synergistic action was detected against these bacterial strains. It was most marked in the combination fosfomycin/nalidixic acid against Serratia marcescens. An acceleration of the fosfomycin action on the bacterial cells and the speed of bacteriolysis was observed in weakly acid medium (pH 6.0), in the presence of 10% active human serum and with increasing glucose-6-phosphate concentrations in the test medium. There is no parallel resistance between gentamicin and fosfomycin against Pseudomonas aeruginosa.     

In vivo and in vitro toxicity of phospholipase C from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces phospholipase C (PLC), a heat-labile hemolysin. Histopathological analysis of PLC-treated mice revealed that the primary target organs involved in PLC-induced toxicity were the liver and kidney. Mice treated i.v. with PLC demonstrated significant tubular epithelial necrosis of the kidney with hematuria, while when given i.p. they exhibited hepatonecrosis with cellular infiltration. Splenomegaly was also a consistent finding. Results from in vitro studies indicate that PLC is toxic for mouse peritoneal cells and human leukocytes.     

磷霉素与氨基糖苷类药物联用对体外铜绿假单胞菌生物被膜形态学的影响

目的:通过研究磷霉素与氨基糖苷类药物(阿米卡星、异帕米星)联合应用对铜绿假单胞菌生物被膜的体外联合效应,探讨磷霉素对氨基糖苷类药物在抑制铜绿假单胞菌生物被膜形成的作用。方法:采用改良的组织培养平板法建立铜绿假单胞菌PAO1生物被膜模型,应用银染法快速鉴定法观察经不同剂量的两类药物单用与联合作用处理后的PAO1生物被膜结构,对其形态学进行描述,并采用计算机图像处理系统进行图像分析。结果:银染法快速鉴定的形态学结果显示,PAO1生物被膜上分布着浓密黑染交织物质,其间隙中聚集着短杆状细菌体;同一剂量下,联药组的黑染交织物较单药组少;磷霉素分别与阿米卡星和异帕米星联用时的抑制被膜形成作用基本相似。计算机图像处理系统的分析显示,平均光密度值随着药物的剂量增大而逐渐减小,联药组的平均光密度值显著低于单药组的平均光密度值(P<0.05)。结论:研究发现,磷霉素与氨基糖苷类药物联用对铜绿假单胞菌生物被膜形成的抑制具有显著增效作用。     

碳青霉烯类抗生素耐药铜绿假单胞菌的体外联合药敏研究

目的评价环丙沙星(CIP)分别与头孢哌酮/舒巴坦(CFS)、哌拉西林/三唑巴坦(TZP)联合用药对临床分离获得的碳青霉烯类抗生素耐药的铜绿假单胞菌(CRPA)的体外抑菌作用。方法采用棋盘法设计,琼脂平板稀释法测定抗菌药物对38株临床分离的CRPA的最低抑菌浓度(MIC),并计算抑菌指数(FIC指数)判断联合抑菌效应。结果 CIP与CFS联用后,5.3%为协同作用,81.5%为相加作用,13.2%为无关作用,没有拮抗作用;CIP与TZP联用后,7.9%为协同作用,73.7%为相加作用,18.4%为无关作用,没有拮抗作用。上述药物联用后,各药MIC50均明显降低,浓度-累积抑菌率曲线均表现为左移。结论 CIP分别与CFS、TZP联用对CRPA体外联合抗菌效应主要表现为协同和相加作用。     

In vitro activity of antibiotic combinations against Pseudomonas aeruginosa biofilm and planktonic cultures

In order to investigate the hypothesis that the combination of clarithromycin with other antibacterial agents offers a successful treatment in the eradication of Pseudomonas aeruginosa biofilm, we first determined the activity of eight antimicrobial agents against planktonic cultures of P. aeruginosa isolates by the microdilution technique. Second, we determined the in vitro effects of these antimicrobial agents individually and in combination against planktonic cultures and pre-formed biofilms of P. aeruginosa PA01. Drug combinations with marked activity were tested on biofilms of clinical isolates. The percentages of planktonic culture survival and biofilm persistence were determined using spectrophotometry and scanning electron microscopy, respectively. Bacterial enumeration was used as a more quantitative method to assess the viability of bacteria in the biofilm. Among the antibacterial agents tested, tobramycin and polymyxin B had the strongest activity against planktonic cultures when tested alone. Synergistic activity was observed for the combination chitosan/tobramycin against planktonic cultures but not biofilms, and for the combination tobramycin/clarithromycin against biofilms but not planktonic cultures. The results suggest that the combination clarithromycin/tobramycin may be successful for eradicating infections involving bacterial biofilms such as in cystic fibrosis patients chronically infected by P. aeruginosa. Further studies on representative isolates in vivo are warranted to support these results.     

In vitro activity of sisomicin, an aminoglycoside antibiotic, against clinical isolates.

Sisomicin, a new aminoglycoside antibiotic which is produced by Micromonospora myoensis, was studied against 565 clinical isolates of gram-negative bacilli and gram-positive cocci. With the exception of Serratia marcescens, over 90% of isolates of gram-negative bacilli were inhibited by 1.56 mug/ml or less of sisomicin. Sisomicin was slightly more active than gentamicin and tobramycin aganist isolates of Escherichia coli, Proteus mirabilis and Klebsiella spp. It was substantially more active than butirosin and kanamycin against all gram-negative bacilli. Isolates of gram-negative bacilli which were resistant to gentamicin and tobramycin were also resistant to sisomicin. Most of these isolates were sensitive to amikacin.     

N-formimidoyl-thienamycin and norfloxacin against multiple-resistant Pseudomonas aeruginosa strains. Combined in vitro activity and comparison with 14 other antibiotics

The in vitro susceptibility of 54 multiple-resistant strains of Pseudomonas aeruginosa to 16 antipseudomonal agents were determined. The majority of these organisms were also beta-lactamase-producing strains. Norfloxacin, N-formimidoyl-thienamycin and aztreonam showed the best antipseudomonal activity, with minimum inhibitory concentrations for 90% of isolates (MIC90) of 2, 8, and 8 mg/l respectively. Of the aminoglycosides, only amikacin (MIC90 = 32 mg/l) showed satisfactory activity, whereas among beta-lactam antibiotics, cefotaxime, ceftriaxone and moxalactam (MIC90 = 32 mg/l) were more active than cefoperazone (MIC90 = 128 mg/l), cefsulodin (MIC90 = 128 mg/l), piperacillin (MIC90 = 512 mg/l) and azlocillin (MIC90 = 1024 mg/l). A synergistic or partially synergistic interaction (fractional inhibitory concentration index less than 1) was demonstrated in vitro against 37% of the strains when N-formimidoyl-thienamycin was combined with norfloxacin.     

蛇葡萄素与8种抗生素联用的体外抗铜绿假单胞菌作用研究

目的:研究蛇葡萄素与8种抗生素联用的体外抗铜绿假单胞菌作用。方法:采用棋盘式微量稀释法测定蛇葡萄素分别与头孢曲松、头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、头孢哌酮、环丙沙星、阿米卡星、哌拉西林、头孢吡肟8种抗生素联用对铜绿假单胞菌(PA)标准菌株ATCC27853和7株临床分离菌株PA135、PA216、PA276、PA281、PA291、PA314、PA319的最小抑菌浓度(MIC),以分级抑菌浓度指数(FICI)评价其联用效果;并以临床分离菌株PA319为目的菌株,设置正常对照组、蛇葡萄素单用组、抗生素单用组以及二者联用组,以联用时蛇葡萄素与抗生素的MIC为作用浓度,计数作用0、4、8、12、24 h的菌落数,绘制时间-杀菌曲线。结果:针对上述8株菌株,蛇葡萄素单用的MIC为128～256 mg/L,分别与头孢曲松、头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、头孢哌酮、环丙沙星、阿米卡星、哌拉西林、头孢吡肟联用时FICI≤1的分别有8、8、7、6、4、4、6、6株。在时间-杀菌曲线中,与抗生素单用比较,蛇葡萄素分别与头孢曲松、头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、头孢哌酮、环丙沙星、阿米卡星、哌拉西林、头孢吡肟联用后菌落数的下降幅度分别为2.65、2.30、0.42、0.47、0.53、1.19、1.74、1.04 lgCFU/mL。结论:蛇葡萄素与头孢曲松、头孢哌酮/舒巴坦联用时对铜绿假单胞菌的抗菌效果更好。     

Immunosuppressive effects of the macrolide antibiotic bafilomycin towards lymphocytes and lymphoid cell lines

The effects of bafilomycin macrolide antibiotics on primary lymphocytes and on tumor cell lines were investigated. Bafilomycin A markedly suppressed DNA, RNA, and protein synthesis in splenocyte cultures of several inbred mouse strains. Bafilomycins were also inhibitory towards cultures of concanavalin A- or lipopolysaccharide-activated murine spleen cells, and inhibited the mitogen-induced differentiation of B lymphocytes into immunoglobulin-secreting plasma cells. Corresponding results were obtained in human cell cultures. A hydrolysis product of the bafilomycin molecule was inactive. Bafilomycin also inhibited the growth of various lymphoid cell lines, the B cell line BCL1, the macrophage cell lines J774 and P338D1, and the T cell line EL4. The sensitivity of the tumor cell lines increased when, simultaneously with bafilomycin, mitogens were applied to the cell cultures. The immunosuppressive action of cyclosporin A could be enhanced by bafilomycin, which could be of importance for the elucidation of the molecular mechanism of T cell suppression, and for applied medical research.