Bactericidal activity of various antibiotics against biofilm-producing Pseudomonas aeruginosa

Clinical isolates of Pseudomonas aeruginosa were collected from hospitals in Tehran, Iran, and identified using biochemical tests. A modified microtitre plate test was used to determine the biofilm-forming capacity of the isolates, measured with an enzyme-linked immunosorbent assay (ELISA) reader. Results showed that P. aeruginosa strain 214 was the most efficient at producing biofilm compared with the other strains. Observation of the bacterial biofilm on Teflon sheets and on a catheter using a scanning electron microscope showed greater biofilm formation on the catheter than on Teflon sheets. In this study, we investigated the bactericidal activity of fluoroquinolones, beta-lactams, macrolides and aminoglycoside. The results showed differences in the antibiotic susceptibility of planktonic and biofilm cell populations. Fluoroquinolones showed more potent activity than the other antibiotics, and biofilms were completely eradicated by treatment with 16 x the minimum inhibitory concentration (MIC) of ciprofloxacin and 64 x MIC of ofloxacin, whereas all biofilms survived 2560 microg/mL of imipenem and ceftazidime. Production of an exopolysaccharide matrix is one of the distinguishing characteristics of biofilms. It has been suggested that this matrix prevents access of antibiotics to the bacterial cells embedded in the community. In this study, we also evaluated the permeation of antibiotics through alginate of P. aeruginosa strain 214 using a sandwich cup method. Macrolides were most efficient, showing 100% penetration; fluoroquinolones and beta-lactams had a high permeation rate > 75%, whereas the rates for aminoglycosides were low (amikacin = 59%; gentamicin = 73%).     

碳青霉烯类抗生素对铜绿假单胞菌抗菌活性的检测

目的 探讨铜绿假单胞菌(PA)临床分离株对碳青霉烯类抗生素耐药性与其产AmpCβ内酰胺酶(AmpC酶)的关系.方法 收集并鉴定PA临床分离株,采用头孢西丁敏感试验对产AmpC酶的菌株进行初筛.运用Kirby-Bauer纸片扩散法、固体培养基连续稀释法检测头孢菌素类与碳素霉烯类抗生素对PA筛选菌株的抗菌活性.采用三维确认试验检测碳青霉烯类抗生素耐药菌株产生AmpC酶的情况.结果 PA筛选菌株对头孢呋肟钠(CXM)、头孢噻甲羧肟(CAZ)、头孢噻肟钠(CTX)、头孢哌酮钠(CFP)及盐酸头孢吡肟(FEP)的耐药率分别为100%、36.3%、63.6%、54.5%、22.7%;对亚胺培南(IPM)、帕尼培南(ETP)、美罗培南(MEM)的耐药率分别为40.9%、45.0%、18.1%,部分菌株对所有试验用药均耐药.IPM、ETP、MEM对PA筛选菌株的最低抑菌浓度(MIC)均≥19 μg/ml,且各试药的最低杀菌浓度(MBC)值与MIC值相差较大,约36.0%菌株的MBC是MIC的16～32倍.约22.6%菌株检测出产生AmpC酶.结论 我市医院近半年来收集的PA对第4代头孢菌素类抗生素FEP具有较高的敏感性;对临床用碳青毒烯类抗生素MEM的敏感性明显高于IPM与ETP,但有较大比例的耐药菌株出现;部分菌株耐药性形成可能与其产生AmpC酶有关系.     

Bactericidal activity of biapenem against various efflux system mutants of Pseudomonas aeruginosa

The bactericidal activity of biapenem, a new carbapenem, against various efflux-mutants of Pseudomonas aeruginosa was compared with those of imipenem, panipenem, meropenem and ceftazidime. The bactericidal activity of biapenem against P. aeruginosa KG5001, a strain deficient in MexAB-OprM, MexCD-OprJ and MexXY-OprM, was very strong compared with those of imipenem and meropenem. In terms of bactericidal activities, biapenem and imipenem had similar activities against P. aeruginosa KG5003, a strain overexpressing MexAB-OprM, as against P. aeruginosa KG5001, however meropenem and ceftazidime had weaker activities against KG5003 than KG5001. The bactericidal activity against P. aeruginosa KG5007, a strain overexpressing MexCD-OprJ, was observed only by biapenem. The bactericidal activity of biapenem was strong and not influenced by all of these three efflux systems.     

Synergistic effect of artocarpin on antibacterial activity of some antibiotics against methicillin-resistant Staphylococcus aureus,Pseudomonas aeruginosa,and Escherichia coli

Context: Antibacterial resistance has dramatically increased and resulted in serious health problems worldwide. One appealing strategy to overcome this resistance problem is the use of combinations of antibacterial compounds to increase their potency.

Objective: The objective of this study is to determine the synergistic effects of artocarpin for ampicillin, norfloxacin, and tetracycline against methicillin-resistant Staphylococcus aureus (MRSA) as well as the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli.

Materials and methods: A broth microdilution method (1.95–250?µg/mL) was used to determine the minimum inhibitory concentration (MIC) of artocarpin and the antibiotics. Any synergistic effects were evaluated at their own MIC using the checkerboard method and a time-kill assay at 37?°C for 24?h.

Results and discussion: Artocarpin showed antibacterial activity against MRSA and E. coli with an MIC value of 62.5?µg/mL, and against P. aeruginosa with an MIC value of 250?µg/mL. The interaction of artocarpin with all tested antibiotics produced synergistic effects against MRSA with a fractional inhibitory concentration index (FICI) of 0.15–0.37. In addition, a combination of artocarpin and norfloxacin showed a synergistic effect against E. coli with an FICI value of 0.37, while the combinations of artocarpin and tetracycline as well as artocarpin and norfloxacin exhibited synergy interactions against P. aeruginosa with FICI values of 0.24 and 0.37, respectively. Time-kill assays indicated that artocarpin enhanced the antimicrobial activities of tetracycline, ampicillin, and norfloxacin against MRSA as well as Gram-negative bacteria.     

Liposome-encapsulated antibiotics: preparation, drug release and antimicrobial activity against Pseudomonas aeruginosa

Ticarcillin- and tobramycin-resistant strains of Pseudomonas aeruginosa were shown to have a markedly increased sensitivity to antibiotics enclosed in liposomes. This was demonstrated by growth inhibition of two resistant P. aeruginosa strains in the presence of the liposome-enclosed ticarcillin and tobramycin at 2 per cent and 20 per cent of their respective minimum inhibitory concentration. The liposome-enclosed antibiotic was as effective against the beta-lactamase-producing strain as against the non-beta-lactamase producing strain. Entrapment efficiency of the two antibiotics with the dehydration-rehydration vesicle (DRV) method was largely superior to other methodologies used. Kinetic studies with DRV demonstrated that tobramycin and ticarcillin-loaded liposomes still contained 83 per cent and 67 per cent of drug respectively after 24 h at 37 degrees C.     

哌拉西林与加替沙星联合对铜绿假单胞菌的联合药敏研究

评价哌拉西林与加替沙星联合用药,对30株临床分离的铜绿假单胞菌的体外联合抗菌效应.采用棋盘法设计,微量肉汤稀释法测定.测定不同浓度组合的抗菌药物对30株临床分离的铜绿假单胞菌的最低抑菌浓度,并计算FIC指数判定联合效应.FIC≤0.5为协同作用,0.5＜FIC≤1为相加作用,1＜FIC≤2为无关作用,FIC＞2为拮抗作用.哌拉西林与加替沙星联合应用后,其MIC50显著降低.FIC指数分布FIC≤0.5占40%;0.5＜FIIC≤1占50%;1＜FIC≤2占10%;FIC＞2为0.结果表明加替沙星与哌拉西林联合用药后,对铜绿假单胞菌基本表现为协同作用和相加作用,并以协同作用为主,无关作用较少,无拮抗作用.     

In-vitro activities of various antibiotics, alone and in combination with amikacin against Pseudomonas aeruginosa.

The in-vitro activities of various antibiotics, either alone or in combination with amikacin were assessed using clinical isolates of Pseudomonas aeruginosa. The minimum inhibitory concentrations (MIC) of these antibiotics were determined by microbroth dilution method against 50 clinical strains. The MIC values showed that 96, 94, and 74% of the isolates were susceptible or moderately susceptible to amikacin, meropenem and ceftazidime, respectively. The in vitro activities of ceftazidime and meropenem in combination with amikacin were determined by microbroth chequerboard technique and results were interpreted using the fractional inhibitory concentration (FIC) index. With a FIC index of ≤0.5 as borderline, synergistic interactions were more frequent with ceftazidime (70.8%) than with meropenem (40%). No antagonism was observed.     

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.     

碳青霉烯类药物对铜绿假单胞菌体外抗菌行为比较

目的:比较不同碳青霉烯类药物对临床分离铜绿假单胞菌体外抗菌行为差异,包括体外抗菌活性以及细菌形态学改变.方法:采用纸片法和标准琼脂稀释法测定临床分离的铜绿假单胞菌对亚胺培南、美洛培南、帕尼培南和头孢他啶的敏感性;电镜观察铜绿假单胞菌在不同浓度的亚胺培南、美洛培南、帕尼培南和头孢他啶中不同时间细菌形态变化.结果:14%的铜绿假单胞菌在美洛培南纸片周围呈"双环"抑菌形态;在MH培基中,帕尼培南的MIC值明显高于亚胺培南和美洛培南,但在MM培养基中,帕尼培南的MIC值是原来的1/8～1/2;亚胺培南和帕尼培南使铜绿假单胞菌细胞呈圆球形改变,头孢他啶使细菌伸长如丝状,低浓度美洛培南的细菌呈纺锤形改变.结论:帕尼培南体外抗菌作用受培养基种类影响大,MH培养基测定结果可能无法准确反映其抗菌活性;美洛培南杀菌作用不完全且诱导细菌呈纺垂体样改变,可能导致内毒素释放增加,影响临床严重感染的预后.     

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.     

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 vitro antimicrobial activity of carbapenem antibiotics against Pseudomonas aeruginosa, measured using a low-concentration Mueller-Hinton Agar culture medium]

Ohya et al. noted that the antibacterial activity of carbapenem-family antibiotics against Pseudomonas aeruginosa was significantly enhanced through lowering the basic amino acid concentration in the culture medium. They reported that there was a marked difference in antimicrobial activity of panipenem (PAPM) against Pseudomonas aeruginosa between the culture medium with Mueller-Hinton Agar (MHA) diluted with distilled water and the non-diluted culture medium. We used 2,312 strains of fresh Pseudomonas aeruginosa, isolated from clinical materials, to examine the antibacterial activity of PAPM in non-diluted and diluted culture media. For testing the susceptibility, we employed the Showa disc method and agar plate dilution method. In the Showa disc method, the inhibition diameters of the tested microbial strains showed a larger distribution for both 16-fold and 40-fold diluted MHA, compared to the non-diluted MHA. The MIC values in the agar plate dilution method were also smaller in distribution for the 16-fold as well as the 40-fold diluted MHA, compared to the non-diluted culture media. Approximately 90% of the strains showed decreased MIC values, 2-8 times in the 16-fold diluted MHA and 2-16 times in the 40-fold diluted MHA. From the above results, we confirmed that the in vitro antibacterial activity of PAPM against Pseudomonas aeruginosa was enhanced through lowering the MHA concentration.     

Inactivation of resistant Pseudomonas aeruginosa by antibacterial combinations

P. aeruginosa resistant to preservative concentrations of benzalkonium chloride, phenylmercuric nitrate and chlorocresol in nutrient broth was inactivated by using phenylethanol-antibacterial combinations. EDTA-antibacterial combinations also showed increased activity using benzalkonium and chlorocresol. P. aeruginosa resistant to 0·25% chlorbutol was inactivated using phenylethanol-chlorbutol combinations at concentrations which were ineffective alone. Similarly P. aeruginosa having an increased resistance to chlorhexidine was inactivated by phenylethanol-chlorhexidine combinations at concentrations that were ineffective alone. Phenylethanol showed a greater general usefulness than EDTA at the concentrations tested.     

Antibacterial activity of liposom e-encapsulated antibiotics against Pseudomonas aeruginosa attached to the matrix of human dermis

The present studies were undertaken to compare the antibacterial activity of liposome vesicles containing amikacin, ciprofloxacin or polymyxin B in the removal of P. aeruginosa organisms from microcolonies growing on sections of the matrix of human dermis. Encapsulation efficiency of antimicrobials inside cationic liposomes was 30% for amikacin, 50% for ciprofloxacin, and 100% for polymyxin B. The sections of dermis were colonized for 72h with P. aeruginosa strains isolated from burn wounds. After that time, an intense growth of microorganisms on the dermis surface was observed. The sessile organisms were treated (with mild shaking) with solutions of either liposomal or free amikacin, ciprofloxacin, and polymyxin B for 1h, and also with a mixture of liposomal or free ciprofloxacin and polymyxin B (1:1) for 20min. After treatment with liposomal antimicrobials, the mean per cent of viable cells attached to the dermis was 48.7% for liposomal amikacin, 17.4% for liposomal ciprofloxacin, 19.1% for liposomal polymyxin B, and 3.6% for a mixture of liposomal ciprofloxacin and liposomal polymyxin B. Removal of P. aeruginosa from microcolonies growing on the dermal matrix was more effective when liposomal formulations were used compared to the free antibiotics. Therefore, cleansing of the contaminated matrix of human dermis with liposomal ciprofloxacin, liposomal polymyxin B or with the mixture of both liposomal antibiotics seems to increase the efficacy at the removal of attached bacterial cells.     

Antibacterial activity of liposome-encapsulated antibiotics against Pseudomonas aeruginosa attached to the matrix of human dermis.

The present studies were undertaken to compare the antibacterial activity of liposome vesicles containing amikacin, ciprofloxacin or polymyxin B in the removal of P. aeruginosa organisms from microcolonies growing on sections of the matrix of human dermis. Encapsulation efficiency of antimicrobials inside cationic liposomes was 30% for amikacin, 50% for ciprofloxacin, and 100% for polymyxin B. The sections of dermis were colonized for 72 h with P. aeruginosa strains isolated from burn wounds. After that time, an intense growth of microorganisms on the dermis surface was observed. The sessile organisms were treated (with mild shaking) with solutions of either liposomal or free amikacin, ciprofloxacin, and polymyxin B for 1 h, and also with a mixture of liposomal or free ciprofloxacin and polymyxin B (1:1) for 20 min. After treatment with liposomal antimicrobials, the mean per cent of viable cells attached to the dermis was 48.7% for liposomal amikacin, 17.4% for liposomal ciprofloxacin, 19.1% for liposomal polymyxin B, and 3.6% for a mixture of liposomal ciprofloxacin and liposomal polymyxin B. Removal of P. aeruginosa from microcolonies growing on the dermal matrix was more effective when liposomal formulations were used compared to the free antibiotics. Therefore, cleansing of the contaminated matrix of human dermis with liposomal ciprofloxacin, liposomal polymyxin B or with the mixture of both liposomal antibiotics seems to increase the efficacy at the removal of attached bacterial cells.     

Structure activity relationships in PIPC-analogues against Pseudomonas aeruginosa

The relationship between the chemical structure and mode of action of piperacillin-analogues (PIPC-analogues) against Pseudomonas aeruginosa was investigated. The antibacterial activities of PIPC-analogues became stronger as the chain length of the alkyl group on the N-4 position in 2,3-dioxopiperazine when tested in constitutively beta-lactamase-producing strain, but not paralleled in wild and beta-lactamase-less strains. The outer membrane permeability was hardly affected by the chain length of the alkyl group at the N-4 position. The stability to beta-lactamase was stronger with the increase of the number of the carbon atoms of N-4 position. In the binding-affinities to the lethal penicillin-binding proteins (PBPs), compounds PIPC (C-2), C-3 and C-4 showed lower ID50 values than compounds C-1, C-6 and C-8. These results suggested that the stability to beta-lactamase was the governing part for the antibacterial activity in constitutively beta-lactamase-producing strain, and the binding affinity to lethal PBPs directly contributed to the antibacterial activity in wild and beta-lactamase-less strains.     

头孢吡肟、头孢哌酮/舒巴坦、亚胺培南对铜绿假单胞菌体外抗菌活性

目的了解铜绿假单胞菌的血清分型及头孢吡肟、头孢哌酮/舒巴坦、亚胺培南对铜绿假单胞菌体外抗菌活性。方法常规分离培养细菌,获纯培养后用VITEK微生物全自动分析仪或API系统鉴定到种;血清分型按试剂盒操作说明进行;药敏实验采用K-B纸片扩散法和肉汤定量稀释法,按NCCLS规定的标准进行;ESBLs检测采用双纸片确认法,AmpC酶检测采用三维实验确认法。结果100株铜绿假单胞菌分型率为83%,B型占50%,G型和E型分别为9%和6%;产ESBLs和产AmpC的阳性率分别为37%、15%;对常用抗菌药物的耐药率分别为左氧氟沙星51%、阿米卡星48%、亚胺培南38%、头孢吡肟33%、头孢他啶20%、头孢哌酮/舒巴坦18%,头孢吡肟、头孢哌酮/舒巴坦、亚胺培南对铜绿假单胞菌的抑菌率分别为59%、43%、15%。结论铜绿假单胞菌的血清型主要以B型为主,次为G型和E型;该菌产酶率和耐药率已相当严重,临床应根据药敏试验情况合理选择使用抗菌药物。     

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.     

用甲基噻唑蓝筛选抗铜绿假单胞菌药物方法的建立

目的:建立适合铜绿假单胞菌的定量、快速、体外高通量药物筛选方法。方法:优化甲基噻唑蓝法检测活细菌数量的实验条件,用MIC方法与MTT法比较2种已知抗生素的抗菌作用。结果:微量培养对细菌生长无影响,最佳MTT浓度和反应时间分别为5 mg·mL-1和1 h。在同一培养时间,菌浓度在1×107～1×1010CFU·mL-1范围内,甲臜的吸收度与菌浓度呈线性相关。用2种抗生素比较MIC方法与MTT法药敏测定的结果一致。结论:MTT微量法进行抗铜绿假单胞菌新药筛选操作简便,省时省力,成本较低,结果稳定,具有可重复性,适宜体外高通量药物筛选。     

The combination effects of antibacterial agents against clinical isolated multiple-drug resistant Pseudomonas aeruginosa

The effectiveness of antibacterial agents against 70 strains of clinically isolated multiple-drug resistant Pseudomonas aeruginosa (MDRP) was measured by the micro dilution method. Fifty of all strains (71%) produced metallo-beta-lactamase and the IMP-1 gene was detected by polymerase chain reaction (PCR). The MIC90 (the minimum inhibitory concentration of an antibiotic necessary to inhibit the growth of 90% of bacterial strains) values of biapenem (BIPM), meropenem (MEPM), tazobactam/piperacillin (TAZ/PIPC), sulbactam/ cefoperazone (SBT/CPZ), cefepime (CFPM), ciprofloxacin (CPFX), pazufloxacin (PZFX), amikacin (AMK) and aztreonam (AZT) were found to be 265, 512, 256, 512, 512, 64, 128, 128 and 128 microg/mL, respectively. The in vitro combination effects of antibacterial agents were examined against 62 strains of MDRP and the synergy or additive effects were evaluated by fractional inhibitory concentration (FIC) index calculated by the checkerboard method. The combination of AMK and AZT showed synergy effects on 15/59 (25.4%) strains of MDRP. The synergy and additive effects on the MDRP strains were also found by the other antibacterial agents combination such as TAZ/PIPC and AMK, CFPM and AMK, and SBT/CPZ and AZT. These results suggested the necessity of further investigation of clinical usefulness.