In vitro activity of vancomycin and teicoplanin againstStaphylococcus aureus andStaphylococcus epidermidis colonizing catheters

In a quantitative in vitro model the activity of vancomycin and teicoplanin in two concentrations (4×MBC and 1 mg/l) againstStaphylococcus aureus and a slime-producingStaphylococcus epidermidis strain colonizing the internal surface of polyurethane and silicone catheters was studied. In comparison with vancomycin, teicoplanin achieved a significantly greater reduction (p<0.05) in the counts ofStaphylococcus aureus andStaphylococcus epidermidis adhering to both polyurethane and silicone catheters.     

In vitro activity of vancomycin against enterococci

The in vitro activity of vancomycin against 40 clinical isolates of enterococci was determined by a macro-tube dilution method and by quantitative killing curve procedures employing the standard medium of our department, i.e. a filtered ox broth. An attempt to remove the influence of technical factors on the MBC determination was made by using an inoculum in the early logarithmic growth phase and ensuring the exposure of all the organisms to the antibiotic. Vancomycin showed a good inhibitory activity for the enterococci tested (MIC90 of 1.6 micrograms/ml, 3.1 micrograms/ml and 1.6 micrograms/ml for S. faecalis, S. faecium and S. durans, respectively), but no bactericidal effect could be demonstrated as measured by the MBCs (greater than 100 micrograms/ml) and killing curve procedures.     

In vitro antimicrobial activity of imipenem in combination with vancomycin or teicoplanin againstStaphylococcus aureus andStaphylococcus epidermidis

The interaction between imipenem and two glycopeptides against staphylococci was examined for potential synergy. Imipenem in combination with vancomycin or teicoplanin exerted a synergistic or additive effect against a majority ofStaphylococcus aureus andStaphylococcus epidermidis isolates tested by the checkerboard method. Synergistic inhibitory effects were frequently accompanied by synergistic bactericidal effects. For a proportion of bacterial isolates of both species, the demonstration of synergy by the checkerboard method was confirmed by time-kill studies using antibiotic combinations at the MICs or at achievable serum antibiotic levels. Only with a single isolate ofStaphylococcus epidermidis was antagonism with either antibiotic combination recorded.     

In vitro and in vivo activities of linezolid alone and combined with vancomycin and imipenem against Staphylococcus aureus with reduced susceptibility to glycopeptides

The objective of this study was to evaluate the in vitro and in vivo efficacies of linezolid (35 mg/kg/5 h), vancomycin (60 mg/kg/5 h), imipenem (30 mg/kg/5 h), linezolid+imipenem, linezolid+vancomycin and vancomycin+imipenem against two clinical Staphylococcus aureus isolates with reduced susceptibility to glycopeptides using time–kill curves and the murine peritonitis model. Time–kill curves were performed over 24 h. For the murine peritonitis model, peritonitis was induced by the intraperitoneal inoculation of 10⁸ CFU/ml of each bacterial strain. Four hours later (0 h), the mice were randomly assigned to a control group or to therapeutic groups receiving subcutaneous treatment for 25 h. Bacterial counts in peritoneal fluid, bacteraemia and mortality rates were determined. The time–kill curves showed that the addition of linezolid to imipenem yielded synergistic results after 24 h. The addition of linezolid decreased vancomycin activity. In the animal model, vancomycin and linezolid monotherapies produced comparable bacterial decreases in mice infected with each strain but linezolid achieved higher rates of blood sterilisation. Linezolid tested either in monotherapy or in combination showed similar efficacy against both strains in terms of bacterial killing, number of negative blood cultures and survival. Linezolid and vancomycin were moderately bactericidal and similar in efficacy against glycopeptide-intermediate or -resistant S. aureus. Linezolid combinations, as effective as linezolid tested alone, could be considered as alternative options for the treatment of glycopeptide-intermediate S. aureus (GISA) infections.     

Comparison of test systems for recognition of methicillin resistance inStaphylococcus aureus

Methicillin resistance inStaphylococcus aureus can be difficult to recognize because it may be expressed in only a small portion of the population. The optimal reference to use in comparing different test systems is controversial. In this study MIC values of 4 mg/l as observed in two experiments, each including 12 test systems (24 h incubation), were used to define strains as methicillin-susceptible. This strict standard was used to compare 24 quantitative and 36 agar diffusion test systems applied to 61 strains. These test systems included six agar media (Mueller Hinton, Oxoid and BBL; and Iso-Sensitest, Oxoid; all with and without 5% NaCl), three broth media of the same type, two incubation temperatures (30 °C and 37 °C), two incubation times (24 h and 48 h) and five disc types [methicillin 10µg (M 10), three types of methicillin 5µg (M 5) and oxacillin 1µg (O 1)]. Standardized high inocula were used. It was concluded that 24 h incubation is preferable to 48 h; at 24 h, differences in recognition of methicillin-resistantStaphylococcus aureus (MRSA) among 12 quantitative test systems were not statistically significant; incubation at 30 °C gave slighty better results in difussion tests. Reproducibility of quantitative data (Friedman test) was better on Mueller Hinton media (p<0.001) than on Iso-Sensitest medium in both dilution and agar diffusion test systems. The rank order of discs was M 5>M 10>O 1 (p<0.001). An inhibition zone of <13 mm with a M 5µg (BBL) disc should be interpreted as MRSA.     

In vitro elution of vancomycin from biodegradable beads.

The current antibiotics delivery system for orthopedic infection treatment uses polymethylmethacrylate (PMMA) beads as a drug release. However the nonbiodegradable nature of the PMMA necessitates a second operation to remove the beads. This article explores the alternative of using biodegradable polymers as antibiotic beads for a long-term drug release. The effect of different processing factors on the release rate of the beads was investigated. To manufacture an antibiotic bead, polylactide-polyglycolide copolymers were mixed with vancomycin. The mixture was compressed and sintered at 55 degrees C to form beads of different sizes. An elution method was employed to characterize the release rate of antibiotic over a 35-day period at 37 degrees C. Biodegradable beads released high concentrations of antibiotic (well above the breakpoint sensitivity concentration) in vitro for the period of time needed to treat bone infection; i.e., 4-6 weeks. A bacterial inhibition test was carried out to determine the relative activity of the released antibiotics. The diameter of the sample inhibition zone ranged from 6.5-10 mm, which is equivalent to 12.5-100% of relative activity. By changing the processing parameters, we were able to control the release rate of the beads. This provides advantages of meeting the specific antibiotics requirement for patients with various surgical infections.     

In vitro parameters of donor-antigen-specific tolerance

Donor-antigen-specific tolerance to the allograft is increasingly considered a reachable goal in the field of transplantation. As our understanding of the processes that govern donor-specific tolerance increases, so must our understanding of ways to detect this state of affairs. Unfortunately, this is not a straightforward procedure, as the mechanisms which govern tolerance are multiple and varied. Previously, the mixed lymphocyte reaction was used as standard to detect unresponsiveness. This approach is not valid for detecting tolerance because it only measures both direct pathway, na?ve and memory responses, whereas the indirect pathway and 'pure' memory responses are more informative parameters for detecting tolerance. Techniques, such as the trans vivo delayed-type hypersensitivity assay, ELISPOT and antigen-specific HLA tetramer analysis address this problem, and the numbers of cell subsets, such as dendritic cells and NKT cells, can also aid us in detecting donor-antigen-specific tolerance.     

In vitro activity of vancomycin and teicoplanin against gram-positive cocci]

The activities of vancomycin and teicoplanin against 148 strains of Gram-positive cocci were tested using agar diffusion and liquid microdilution MIC determination. Tested strains included 84 staphylococci, 32 S. aureus, 52 coagulase-negative staphylococci (CNS), 52 enterococci, and 12 streptococci. Most strains (136) were susceptible to both agents, with inhibition diameters of 17 mm or more. MRSA strains exhibited lower geometric MIC means with teicoplanin (0.90 micrograms/ml) than with vancomycin (1.79 micrograms/ml); this difference was found for methicillin-susceptible S. aureus strains (1.07 and 1.38 micrograms/ml for teicoplanin and vancomycin, respectively). In contrast, methicillin-susceptible and methicillin-resistant strains of CNS exhibited similar MICs (1.60 micrograms/ml approximately). Enterococci were more susceptible to teicoplanin (MIC 0.25 micrograms/ml) than to vancomycin (MIC 1.35 micrograms/ml). Both vancomycin and teicoplanin were thus found to be consistently effective against Gram-positive cocci; however, teicoplanin proved more effective than vancomycin against enterococci and methicillin-resistant S. aureus strains and may therefore be a valuable therapeutic alternative for these multiresistant organisms.     

Role of insertion elements and yycFG in the development of decreased susceptibility to vancomycin in Staphylococcus aureus

The glycopeptide antibiotic vancomycin acts by binding to the D-alanyl-D-alanine terminus of the cell wall precursor lipid II in the cytoplasmic membrane. The purpose of this study was the identification of genes that might be involved in the vancomycin resistance mechanism. To this end, the expression profiles of two vancomycin intermediately resistant Staphylococcus aureus (VISA) strains, the clinical isolate S. aureus SA137/93A (Etest: 8 microg/ml) and its laboratory mutant S. aureus SA137/93G (Etest: 12 microg/ml) were analyzed using an S. aureus full-genome chip. The results indicated that an essential two-component regulatory system, yycF (vicR) and yycG (vicK) was drastically up-regulated in strain SA137/93A. Sequencing of the yycFG promoter region of strain SA137/93A revealed an insertion of IS256 in the predicted promoter region creating a potentially stronger hybrid promoter. In strain SA137/93G, IS256 was not integrated in the yycFG promoter region but, in previous studies, a copy of IS256 had been found to inactivate the tcaA gene (Maki et al. Antimicrob. Agents and Chemother. 48, 1953-1959 (2004)). Detailed population analyses showed that, in addition to the loss of SCCmec, the inactivation of tcaA seems to cause at least part of the increase in teicoplanin and vancomycin resistance in strain SA137/93G.     

In vitro selection of resistance to vancomycin in bloodstream isolates ofStaphylococcus haemolyticus andStaphylococcus epidermidis

The purpose of the study was to determine whether vancomycin-resistant strains ofStaphylococcus haemolyticus could be selected regardless of the initial MIC of vancomycin. Twenty-one bloodstream isolates ofStaphylococcus haemolyticus were studied by broth and agar selection methods. The broth method selected strains for which MICs of vancomycin ranged from 4 to 32 µg/ml and MBCs from 16 to>128 µg/ml. The agar method selected strains for which MICs ranged from 8 to 32 µg/ml and MBCs from 8 to>128 µg/ml. For comparison, seven strains ofStaphylococcus epidermidis were evaluated by the agar selection method. Final MICs of vancomycin ranged from 8 to 16 µg/ml; MBCs ranged from 16 to 64 µg/ml. Clearly, in vitro exposure to vancomycin can select strains ofStaphylococcus haemolyticus andStaphylococcus epidermidis for which MIC values are beyond the susceptible breakpoint.     

Telavancin shows superior activity to vancomycin with multidrug-resistant Staphylococcus aureus in a range of in vitro biofilm models

The activity of telavancin was compared with vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) in planktonic culture and biofilms grown using a range of in vitro models. Antibiotic efficacy was determined using 24 clinical isolates, including healthcare-associated (HA)-MRSA, community-associated (CA)-MRSA and isolates with reduced (intermediate) susceptibility to vancomycin (VISA). Activity against biofilms was compared using three models: 96-peg plates, 96-well flat-bottom plates and a flow-cell system. Cell death was evaluated using a metabolic dye and Live/Dead staining. The planktonic minimum inhibitory concentration (MIC) range for telavancin was lower than that for vancomycin (0.06–0.25 mg/l and 0.5–8 mg/l, respectively). Vancomycin (100?×?MIC) killed, on average, 59 % of cells in HA-MRSA biofilms grown on 96-peg plates, 44 % of cells in CA-MRSA biofilms and 26 % of cells in VISA biofilms. Telavancin (100?×?MIC) killed, on average, 63 %, 49 % and 41 % of cells, respectively. The antibiotics showed similar efficacy against MRSA biofilms but telavancin was more effective against those formed by VISA isolates. In the flow-cell system, antibiotic cell killing was enhanced with both antibiotics, killing up to 80 % of biofilm-associated cells. The variance in cell killing displayed when biofilms were grown using different systems highlights the importance of selecting an appropriate model for antimicrobial efficacy tests. The flow-cell system more closely reflects conditions encountered during infection and is possibly more clinically relevant than a 96-well plate system. Despite differences between the models evaluated, telavancin typically demonstrated improved efficacy over vancomycin, indicating the potential value of the agent in the treatment of biofilm-mediated infections caused by S. aureus, especially multidrug-resistant isolates.     

In vitro stability of polyether and polycarbonate urethanes

The in vitro structural stability of poly-ether-urethanes (PEUs) and poly-carbonate-urethanes (PCUs) was examined under strong acidic (HNO3) or alkaline (NaClO) oxidative conditions and in presence of a constant strain state. Polyurethane (PU) samples were represented by sheets solvent-cast from commercial pellets or by tubular specimens cut from commercial catheters. The specimens were strained at 100% uniaxial elongation over appropriate extension devices and completely immersed into the oxidative solutions at 50 degrees C for 7-14 days. The changes induced by the oxidative treatments were then evaluated by molecular weight analysis, tensile mechanical tests, and scanning electron microscopy. In the experiments with solvent-cast samples, the PEU Pellethane was degraded more in the alkaline oxidative conditions and mainly in the absence of an applied uniaxial stress. All the tested PCUs were, on the contrary, more affected by the acidic oxidative agent. All the PCUs proved to have overall better stability than the PEU. The susceptibility to oxidation was also dependent on the shape and bulk/surface organisation acquired by the same polymer during its processing. When the oxidative test was applied to catheters made of a PEU and a PCU, the results confirmed the better stability of poly-carbonate-urethanes.     

In vitro enzymatic stability of dendritic peptides

PEGylated polyamidoamine (PAMAM) dendrimers as drug carriers have been a topic of interest because of their biomedically favorable features, including minimal toxicity, reduced immunogenicity, and excellent solubility in aqueous and most organic solutions. A PEG shell on dendrimer surface may provide steric hindrance, known as stealth properties of PEG, to stabilize drug molecules to be delivered. In this article, the effects of PEG and coupling sequence of drug, PEG, and dendrimer in modulating the stability of delivered drug molecules were evaluated. N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide was chosen as a model peptide. Dendritic peptides, that is, peptide-dendrimer, peptide-PAMAM-PEG, and peptide-PEG-dendrimer, were constructed based on Starbursttrade mark G3.0 PAMAM dendrimer and characterized by (1)H-NMR spectroscopy. Hydrolysis of dendritic peptides was catalyzed by alpha-chymotrypsin in pH 7.4 PBS buffer containing 5% DMF (v/v) at room temperature. The enzymatic stability of dendritic peptides was peptide-PAMAM-PEG > peptide-PAMAM > free peptide > peptide-PEG-PAMAM. The ratio of PEG/peptide could be reduced for increasing peptide loading while maintaining the delivered peptides' relatively high enzymatic stability. The quantitative analysis of dendritic peptide/enzyme interactions provided the understandings of the molecular structure/stability relationships of dendrimer/drug for the design of an optimal PEGylated dendrimer-based drug-delivery system.