Comparative study of in vitro methods to analyse the antifungal activity of propolis against yeasts isolated from patients with superficial mycoses

AIM: To test a total of 15 strains belonging to four species of yeasts by different in vitro methods against propolis and itraconazole (ITC). METHODS AND RESULTS: Three methods were compared for susceptibility testing of yeast isolates to propolis: disc diffusion method, agar dilution method and National Committee for Clinical Laboratory Standards (NCCLS, M27A) broth microdilution method. ITC was selected as the antifungal agent for comparison study. Using the broth microdilution method, the geometric mean for MIC (microg ml(-1)) with regard to all isolates was < or =0.06 for propolis and < or =0.35 for ITC. The broth microdilution and the agar dilution methods were in good agreement (75%) for propolis against yeasts isolated from patients with superficial mycoses. Using the diffusion method, all strains showed a broad zone of inhibition at the first available reading time (24 or 48 h). An increase of MIC values was accompanied by a decrease of growth inhibition zone diameter. A favourable correlation was found between MIC and inhibition zone around the disc for propolis sample and the correlation coefficient was: r = -0.626 (P < 0.01). CONCLUSIONS: This study suggests the potential value of the agar dilution and disc diffusion method as a convenient alternative method for testing of yeasts to propolis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that propolis and ITC were very active against yeasts from patients with superficial mycoses. The other prominent finding in this study is that RPMI 1640 with L-glutamine was the available broth for the in vitro susceptibility testing of yeasts.     

五环三萜类柴胡皂苷单体对白念珠菌伊曲康唑耐药株活性研究

目的 从柴胡中提取具有抗真菌活性的五环三萜类单体Bp3,研究该单体对伊曲康唑耐药白念珠菌菌株的作用及其与伊曲康唑的相互作用.方法 参照CLSI标准,采用棋盘微量稀释法,测定单用Bp3、伊曲康唑及两者联合使用时对20株伊曲康唑耐药白念珠菌菌株的MIC,计算部分抑菌浓度指数(FIC),判定两药相互作用.结果 单独用药时Bp3及伊曲康唑对伊曲康唑耐药白念珠菌菌株MIC的几何均数值(GM值)分别为1.941 μg/mL和1.008 μg/mL.联合用药时Bp3和伊曲康唑的GM值分别降低为1.189 μg/mL和0.346 μg/mL.2种药物单用和联合使用时MIC值差异均有统计学意义(P＜0.05),联用时在20株耐药株均表现为协同或相加作用.结论 五环三萜类单体Bp3对白念珠菌伊曲康唑耐药株有一定的抑制作用,且与伊曲康唑有协同或相加作用.     

亚胺培南对铜绿假单胞菌敏感和耐药菌株DNA释放的影响

目的：观察亚胺培南对铜绿假单胞菌标准菌株和耐药菌株体外培养释放DNA的影响。方法：选择铜绿假单胞菌的标准菌株和临床分离耐药菌株作为实验菌株,检测其亚胺培南的最低抑菌浓度（MIC）,琼脂糖凝胶电泳法观察不同浓度亚胺培南作用下铜绿假单胞菌的DNA释放情况,并用Quantity One软件分析所释放DNA片段的分子量和含量。结果：亚胺培南作用下铜绿假单胞菌从1／2MIC开始释放小片段DNA,大于1／2MIC仅释放l条大片段DNA,且含量少,等于1／2MIC可释放3条DNA,但无小片段DNA,小于1／2MIC可释放4．5条DNA,且含量多。1／16MIC时,标准菌株释放的第1、5条DNA片段含量偏少,3、4条DNA片段含量偏多,而耐药菌株相反。耐药菌株与标准菌株相比释放的DNA片段有所不同,耐药菌株比标准菌株多释放一条DNA片段（第2条）,分子量为2784bp,耐药菌株的第1、3、4、5条DNA片段分子量均比标准菌株小。耐药菌株释放的第3、4、5条DNA含量明显高于标准菌株,P〈0．01,其统计有显著学意义。结论：不同亚胺培南浓度作用下,铜绿假单胞菌会释放不同片段大小和含量的DNA分子,耐药菌株与标准菌株释放的DNA片段数量和含量有明显差异,其与耐药机制的关系值得进一步研究。     

Broth dilution and disc diffusion methods in the susceptibility testing of pathogenic Candida albicans against four antimycotics

Susceptibility of Candida albicans isolated from mouthrinse specimens during episodes of acute pseudomembranous candidiasis in patients with haematological malignancies was tested by the broth dilution and disc diffusion methods using 24 and 48 h of incubation. The time factor did not significantly affect the results with 5-fluorocytosine. With amphotericin B, prolonged incubation doubled the geometric mean MIC of C. albicans as well as the number of isolates with intermediate sensitivity. With the shorter incubation in the disc diffusion assay, few isolates showed lowered sensitivity to clotrimazole; at 48 h, however, this figure was as high as 54%. The yeasts were highly sensitive to ketoconazole at 24 h, whereas at 48 h the results were bizarre. At 24 h, correlations between disc diffusion and broth dilution methods were satisfactory, with clotrimazole and ketoconazole accounting for most of the discordancies. Accordingly, the disc diffusion results should be recorded at 24 h.     

Development of a method for the quantitative assessment of the microorganism sensitivity to antibiotics by using discs. A study of the patterns of doxycycline diffusion from discs into the nutrient agar]

Doxycycline concentrations provided by the antibiotic diffusion from paper discs into sterile agarized medium and the medium plated with staphylococci and Coli bacteria were studied at different distances from the disc center. Infection of the nutrient medium with the test cultures had no effect on the antibiotic diffusion rate. A linear relation between the logarithm of the antibiotic concentration in the agar and distance from the disc centre were found. Probably it is possible to determine the MIC of the antibiotic with respect to various microorganisms by the value of the radius of the growth inhibition zone around the disc using diagrams expressing such a relation.     

Sensitivity of Certain Porcine and Bovine Mycoplasmas to Antimicrobial Agents in a Liquid Medium Test Compared to a Disc Assay

The sensitivity of some porcine and bovine mycoplasmas to potent antimicrobial agents was examined. Minimal inhibitory concentration (MIC) values were estimated for M. hyosynoviae, M. hyopneumoniae, M. dispar and M. bovis against enrofloxacin, lincomycin, tetracycline, tiamulin and tylosin, in a liquid medium test and in a disc assay. All 6 examined strains of each species and the respective type strains were significantly inhibited. The greatest sensitivity was noted for tiamulin against strains of M. hyosynoviae with a final MIC50 broth value of 0.025 µg ml−1 and disc value of 0.03 µg per disc. Enrofloxacin was found very potent against M. hyopneumoniae with a final MIC50 of 0.025 µg ml−1 and 0.1 µg per disc, and for M. dispar with 0.05 µg ml−1 and 0.03 µg per disc. Most disc assay estimates in ug per disc were similar to or moderately greater than corresponding final broth figures in µg ml−1. It may be possible to convert observed disc assay values into representative final broth MIC values for use in the clinic.     

玫烟色拟青霉适温性及抗多菌灵特性的种内变异

在15 ℃～35 ℃的温度范围内比较测定了玫烟色拟青霉(Paecilomyces fumosoroseus) 6株野生菌的菌落生长、产孢量及孢子活力.结果显示,3个指标均以25 ℃左右最好,但同一温度下菌株间或同一菌株在不同温度下的差异显著, 菌株Pfr116和Pfr6206具有相对稳定优良的生长、产孢及活孢率性状.对不同浓度多菌灵对6株野生菌的菌落生长和单孢菌落形成的影响测定表明,菌株间存在较大差异.不同多菌灵浓度对单孢菌落形成的抑制率观察值可用逻辑斯蒂模型较好地拟合(r²≥0.90).用所获参数估计的最低禁菌浓度MIC值显示,Pfr4205和Pfr116对多菌灵表现为低抗性（MIC≤20.0 μg·ml^-1）;Pfr153、Pfr612和Pfr2175属于低水平中抗,MIC值仅略大于20 μg·ml^-1;而Pfr6206的MIC值高达93.5 μg·ml^-1,接近MIC>100 μg·ml^-1的高抗标准.因此,Pfr6206为适温性和抗多菌灵特性俱佳的菌株,可作为抗多菌灵和适应不同季节或地区的害虫生防菌剂的候选菌株.     

Pharmacodynamic parameters and hydrophobicity changes induced by meropenem in Acinetobacter baumannii.

The suppression of bacterial growth of four Acinetobacter baumannii strains after 60 min exposure to meropenem at supra-inhibitory concentrations (postantibiotic effect; PAE) or at supra-subinhibitory concentrations (postantibiotic-sub-MIC effect; PA SME) was studied. The duration of the PAE was dependent on antibiotic concentration and on the strain. Meropenem at 2x or 4x the minimum inhibitory concentration (MIC), with the exception of one strain treated with 4x MIC, did not provoke suppression of bacterial growth compared with untreated controls. The highest concentration of meropenem (8x MIC) induced PAE for the strains tested in the range of 0.6-6.9 h. The effect of supra-subinhibitory concentrations of meropenem (2x, 4x or 8x MIC + 0.2x MIC) on bacterial growth was more efficient compared with supra-inhibitory concentrations alone. Two out of the four strains treated did not renew their growth. Bacterial suspensions exposed to meropenem showed reduced surface hydrophobicity. Decreases in hydrophobicity were associated with longer PAE and PA SME depending on the strain.     

In vitro activity of cefepime against ESBL-positive clinical strains of gram-negative rods

The aim of this study was to determine an in vitro activity of cefepime against ESBL-positive clinical strains of Gram-negative rods isolated from hospitalized patients. Experiments were performed with 100 ESBL-positive strains of Gram-negative rods isolated from clinical samples in 2004. Strains were identified with the use of automatic ATB Expression system and biochemical ID 32 GN tests (bioMdrieux sa). Extended-spectrum beta-lactamases (ESBLs) were detected by means of disc diffusion methods: the double-disc synergy test (DDST) and the diagnostic disc test (DD, Oxoid Ltd, UK). Susceptibility in vitro of ESBL producers to 4th generation--cefepime was determined with gradient diffusion method Etest (AB Biodisk, Solna, Sweden). MIC value of cefepime was assessed for each strain. Among 100 ESBL-producing strains, 94--belonged to enteric rods and 6--to nonfermentative rods. The greatest number of strains belonged to the species Serratia marcescens (27% of all strains) and next--to the species Enterobacter cloacae (21%). Fourteen strains were susceptible (S) in vitro to cefepime, 12--intermediately susceptible (I) and 74--resistant (R). Application of cefepime in a therapy of infections caused by ESBL-positive strains of Gram-negative rods highly susceptible in vitro to this antibiotic, should be considered.     

Occurrence of high-level mupirocin resistant coagulase-negative staphylococci in hospitals and their characterization

Occurrence of high-level mupirocin resistant coagulase-negative staphylococci in 5 hospitals in the region of Gdansk was determined. The study was carried out on 192 staphylococcal strains isolated from patients and medical staff. The mupirocin resistant strains were detected by 5 microgram mupirocin disc. The minimal inhibitory concentration (MIC) for mupirocin was estimated by E-tests. The mupirocin resistant strains were characterised by antibiotic sensitivity, including MIC for glycopeptides and oxacillin. Biotypes of resistant strains were also determined. Eight high-level mupirocin resistant strains (4.7%) were found. Only one strain expressed low-level resistance. All but one of high-level mupirocin resistant strains were resistant to methicillin. Six of them belonged to the S. epidermidis species but differences in the biotypes and antibiotic resistance patterns of these strains suggest they did not have a common origin.     

Sanfetrinem, the member of trinems--in vitro activity against Klebsiella pneumoniae producing ESBL

Sanfetrinem is the first member of tricyclic beta-lactams (trinems) which can be administered orally as a hexatil ester. His chemical structure is related to carbapenems. High stability to many beta-lactamases and human renal dehydropeptydase were described. The investigation was performed on 43 strains of Klebsiella pneumoniae producing ESBL isolated from hospitalized patients. The MICs of sanfetrinem and imipenem were determined by E-test. Additionaly, susceptibility to antibiotics was tested by disc diffusion method. Klebsiella pneumoniae ATCC 700603, Escherichia coli ATCC 25922 and Escherichia coli ATCC 35218 were used as a control strains. MIC50 and MIC90 of sanfetrinem and imipenem amounted respectively 0,38/3 mg/1 and 0,19/0,25 mg/l. Range of MIC value was from 0,064 mg/l to 4 mg/l for sanfetrinem, and from 0,094 mg/l to 0,38 mg/l for imipenem. Additionaly, geometric and aritmetic means were calculated to both antibiotics. All results of study were compared using correlation factor and "Student" t-test. None of these 43 Klebsiella pneumoniae strains was resistant to imipenem and cefepime. Majority of isolates demonstrated susceptibility to ciprofloxacin and cefoxitin--90,7% and 74,4% respectively All strains were resistant to gentamicin, amikacin and trimethoprim/sulfamethoxazole.     

Influence of the postantibiotic effect and postantibiotic Sub-MICs effect of netilmicin,tobramycin, ciprofloxacin and pefloxacin on alginate production byPseudomonas aeruginosa

The postantibiotic effect (PAE) (postantibiotic phase induced by 2× or 4×MIC) as well as the postantibiotic effect of subinhibitory concentrations (0.1×, 0.2× and 0.3× MIC) (PA SME) of netilmicin, tobramycin, ciprofloxacin and pefloxacin affected the production of the virulence factor alginate by aP. aeruginosa strain. Aminoglycosides and ciprofloxacin at a concentration of 4× MIC inhibited the alginate production more significantly than 2× MIC. Suprainhibitory concentrations of aminoglycosides were more effective than pefloxacin (2× or 4× MIC) and ciprofloxacin (2× MIC). PA SME demonstrated by the above antibiotics (with the exception of ciprofloxacin 2× MIC +00.1× MIC) suppressed alginate production more efficiently.     

Antimicrobial potentiality of a new non-antibiotic: the cardiovascular drug oxyfedrine hydrochloride

Ten cardiovascular drugs, having diverse pharmacological action, were screened for possible antimicrobial property against known eight sensitive bacteria, belonging to Gram positive and Gram negative types. Although five drugs failed to show antimicrobial activity and three had moderate antimicrobial action, oxyfedrine HCl and dobutamine were seen to possess pronounced antimicrobial property. Oxyfedrine was further tested in vitro against 471 strains of bacteria from two Gram positive and fourteen Gram negative genera. The minimum inhibitory concentration (MIC) of oxyfedrine was determined by agar dilution method, which ranged from 50-200 microg/ml in most of the strains, while some strains were inhibited at even lower concentrations. In animal experiments, this compound was capable of offering significant protection to Swiss strain of white mice, challenged with 50 median lethal dose (MLD) of a virulent strain of Salmonella typhimurium at concentrations of 15, 30 and 60 microg/mouse. The in vivo results were highly significant according to chi-square test.     

Postantibiotic effect of various antibiotics on Legionella pneumophila strains isolated from water systems

The postantibiotic effects (PAE) of azithromycin, clarithromycin, ciprofloxacin, and levofloxacin were investigated against Legionella pneumophila (L. pneumophila) strains isolated from several hot water systems of different buildings in Istanbul. Each strain in logarithmic phase of growth was exposed to concentrations of antibiotics equal to minimum inhibitory concentration (MIC) and 4× MIC for 1?h. Recovery periods of test cultures were evaluated after centrifugation using the viable counting method. The mean values of PAEs for the strains of L. pneumophila, azithromycin at a concentration equal to and 4 times of MIC values were found 1.75?±?0.28 h and 4.06?±?0.44?h, for clarithromycin 2.98?±?0.70?h and 4.18?±?0.95?h, for ciprofloxacin 2.97?±?0.63?h and 4.70?±?0.63?h, for levofloxacin 2.05?±?0.33?h and 3.78?±?0.46?h, respectively. All of the antibiotics showed increased PAE values in a concentration-dependent manner. The findings of our study may play useful role in selecting the appropriate timing of doses during therapy with antimicrobials to treat patients infected with L. pneumophila.     

Postantibiotic effects of gentamicin and netilmicin onSerratia marcescens: Effects on hydrophobicity and motility

The impact of postantibiotic effect (PAE) of aminoglycosides (gentamicin, netilmicin) on cell-surface hydrophobicity and motility of a clinical isolateSerratia marcescens was evaluated. For the induction of PAE 2× and 4×MIC concentrations of both antibiotics were used. Gentamicin and netilmicin induced a PAE of similar duration after 2×MIC concentration (2.7 and 2.8 h, respectively). Both aminoglycosides demonstrated concentration-dependent PAE. At a concentration of 4×MIC they produced PAEs of 5.9 and 8.2h, respectively. The evaluation of hydrophobic properties ofS. marcescens after affecting PAE showed that both aminoglycosides inhibited adherence to xylene. This inhibition was also concentration-dependent. More expressive, was netilmicin which inhibited the adhesion by 70.5% at 2×MIC and by 85.2% at 4×MIC. Netilmicin inhibited also the adhesion to nitrocellulose filter by 34.7% at 4×MIC. Exposure of the bacterial cells to suprainhibitory concentrations of both aminoglycosides resulted only in moderate inhibition of motility of strain tested compared to the unexposed cells.     

Postantibiotic effects and postantibiotic sub-MIC effects of ciprofloxacin, pefloxacin and amikacin on the biological properties ofSalmonella strains

The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PASME) of ciprofloxacin, pefloxacin and amikacin were studied forSalmonella typhimurium andS. enteritidis strains. PAE was induced by 2× and 4×MIC of antibiotics studied for 0.5 h. After PAE and PASME their effect on prophage induction of a lysogenicS. typhimurium strain and on Congo red binding for both strains as a marker of their surface hydrophobicity was examined. The longest PAE was found after treatment with ciprofloxacin, higher values being observed withS. typhimurium. PAEs of pefloxacin and amikacin were much lower, except for the suprainhibitory concentration 4×MIC of amikacin withS. enteritidis (6.9 h). PASMEs of ciprofloxacin did not allow any regrowth of either strain. For other antibiotics the PASME's were different while concentrations of 2×MIC+0.2×MIC and 0.3×MIC, and of 4×MIC+0.1×MIC, 0.2×MIC and 0.3×MIC of amikacin did not allow any regrowth ofS. enteritidis. PAEs of the antibiotics tested did not affect the Congo red binding by bothSalmonella strains, but the PAEs of ciprofloxacin and pefloxacin expressively induced a prophage of lysogenicS. typhimurium strain. We noted the influence of Congo red binding after applying 4×MIC+0.1×MIC, 0.2×MIC and 0.3×MIC of amikacin forS. typhinurium and 2×MIC+0.1×MIC forS. enteritidis.     

Inhibition ofPseudomonas aeruginosa alginate expression by subinhibitory concentrations of antibiotics

The effects of subinhibitory concentrations (1/4, 1/8, 1/16 of the MIC) of quinolones (ciprofloxacin, enoxacin, nalidixic acid, norfloxacin, ofloxacin, pefloxacin), aminoglycosides (amikacin, gentamicin, netilmicin, streptomycin, tobramycin), β-lactams (aztreonam, ceftazidime, imipenem, ticarcilin) and macrolides (erythromycin, roxitromycin) on the excretion of alginate by aP. aeruginosa strain were studied. Both β-lactam and macrolide antibiotics were found ineffective at the concentrations tested, except erythromycin and imipenem at 1/4 MIC. Aminoglycosides at a concentration of 1/4 MIC reduced most effectively the excretion of alginate. Quinolones were also effective at this sub-MIC; 1/16 MIC was ineffective with all antibiotics or stimulated the production of alginate.     

Synergistic activity of melittin with mupirocin: A study against methicillin-resistant S. Aureus (MRSA) and methicillin-susceptible S. Aureus (MSSA) isolates

Methicillin-Resistant Staphylococcus aureus (MRSA) biofilms are involved in various nosocomial infections, being in the limelight of academic research. The current study aimed to determine the antimicrobial effects of melittin on planktonic and biofilm forms of S. aureus. Following the identification of MRSA and SCCmec types (using PCR method), Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), and fractional inhibitory concentration index (FICi), for melittin and mupirocin were determined by broth microdilution assay. Melittin anti-biofilm activity was determined, using a microtiter-plate test (MtP) and scanning electron microscope (SEM) methods. The quorum sensing inhibitory activity of ½ MIC melittin was examined using a quantitative real-time RT-PCR method, and melittin cytotoxicity on Vero cells was examined by tetrazolium-based colorimetric (MTT) test. The Results of our study showed that Geometric means of MIC values of the melittin and mupirocin were 4.4 and 14.22 μg/ml respectively. The geometric mean of the FICi for both melittin-mupirocin was 0.75. No S. aureus biofilm was formed and hld gene (as a biofilm regulator) expression down-regulated. It seems that melittin can be useful in the treatment of S. aureus infections (especially MRSA) by reducing the hld expression. Furthermore, synergistic growth-inhibitory effects of mupirocin with melittin could be considered as a promising approach in the treatment of MRSA isolates.     

Detection and prevalence of pneumococci with increased resistance to penicillin.

Susceptibility to penicillin was determined for 6000 strains of pneumococci isolated during 1974--76 from patients in Alberta and the adjacent region of the Northwest Territories. Strains were considered to be relatively resistant if the minimum inhibitory concentration (MIC) of penicillin was 0.16 microgram (0.26 U)/mL or more, which is eight or more times greater than the MIC for fully susceptible strains. Resistance was detected in 143 strains (2.4%) isolated from 122 patients and belonging to four capsular types. The MIC of the most resistant strains was 0.32 microgram (0.53 U/mL. Penicillin-resistant strains were highly resistant to oxacillin, the MIC being at least 30 times greater than that for penicillin-susceptible strains. Pneumococci resistant to penicillin may readily be detected by the narrowness or absence of a zone of inhibition around a 1-microgram oxacillin disc in susceptibility tests on blood agar. The degree of resistance reported here is relative and does not necessarily preclude successful treatment with full therapeutic doses of penicillin G, but penicillin preparations that give low blood concentrations may not be suitable for treating infections caused by these strains.     

Susceptibility of the Lyme disease spirochete to seven antimicrobial agents.

The antimicrobial susceptibility of five Lyme disease spirochete strains (two human and three tick isolates) was determined. A macrodilution broth technique was used to determine on three separate test occasions the minimal inhibitory concentrations (MICs) of seven antibiotics. The Lyme disease spirochete was most susceptible to erythromycin with a MIC of less than or equal to 0.06 micrograms/ml. The spirochete was also found to be susceptible to minocycline, ampicillin, doxycycline, and tetracycline-HCL with respective mean MICs of less than or equal to 0.13, less than or equal to 0.25, less than or equal to 0.63, and less than or equal to 0.79 micrograms/ml. The spirochete was moderately susceptible to penicillin G with a mean MIC of 0.93 micrograms/ml. All strains were resistant to rifampin at the highest concentration tested (16.0 micrograms/ml).