Antibiofilm effects of amoxicillin-clavulanic acid and levofloxacin in patients with chronic rhinosinusitis with nasal polyposis.

PURPOSE: Microbial biofilms have been implicated in the pathogenesis of chronic rhinosinusitis with nasal polyposis (CRSwNP). The aim of our study was to evaluate in vitro effects of amoxicillin-clavulanic acid and levofloxacin on biofilm formation by bacterial species isolated from sinus tissue in patients with CRSwNP. METHODS: The sinus mucosal specimens were harvested from the upper parts and roof of ethmoid cavity of 48 patients with CRSwNP. Each sample was washed thoroughly in three separate beakers of sterile saline to remove any planktonic bacteria and further subjected to microbiology analysis. The biofilm-forming capacity of isolated strains was detected by microtiter-plate method and the effects of subinhibitory (1/2× to 1/16× MIC) and suprainhibitory concentrations (4, 8, 16, 32, and 64 µg/ml) of amoxicillin-clavulanic acid and levofloxacin on biofilm production were investigated. RESULTS: Bacterial strains were isolated in 42 (87.5%) patients: one microorganism in 80.9% and two microorganisms in 19.1% of patients. The most prevalent bacteria in CRSwNP biofilms were Staphylococcus epidermidis (34%) and S. aureus (28%) followed by S. haemolyticus (12%), Pseudomonas aeruginosa (8%), Moraxella catarrhalis (6%), Streptococcus pneumoniae (6%), and other staphylococci (6%). Subinhibitory concentrations of amoxicillin-clavulanic acid and levofloxacin significantly reduced biofilm formation (p < 0.01 and p < 0.05, respectively), with better efficacy of amoxicillin-clavulanic acid (1/2-1/8× MIC) on staphylococci and levofloxacin (1/2- 1/4× MIC) on M. catarrhalis and P. aeruginosa biofilm formation. Suprainhibitory concentrations of both tested antibiotics (4-64 µg/ml) significantly eradicated mature biofilms of staphylococci (p < 0.01). The effect of levofloxacin on eradication of staphylococcal biofilms was more noticeable, compared to the effect of amoxicillin-clavulanic acid (p < 0.01). Suprainhibitory concentrations of both tested antibiotics had no effect on eradication of previously formed M. catarrhalis and P. aeruginosa biofilms (p > 0.05). CONCLUSIONS: The amoxicillin-clavulanic acid and levofloxacin are shown to be potent antibiofilm agents in patients with CRSwNP. The effects of tested compounds depend on bacterial species and the volume of formed biofilm.

Characterization of bonded stationary phase performance as a function of qualitative and quantitative chromatographic factors in chaotropic chromatography with risperidone and its impurities as model substances.

Numerous stationary phases have been developed with the aim to provide desired performances during chromatographic analysis of the basic solutes in their protonated form. In this work, the procedure for the characterization of bonded stationary phase performance, when both qualitative and quantitative chromatographic factors were varied in chaotropic chromatography, was proposed. Risperidone and its three impurities were selected as model substances, while acetonitrile content in the mobile phase (20-30%), the pH of the aqueous phase (3.00-5.00), the content of chaotropic agents in the aqueous phase (10-100 mM), type of chaotropic agent (NaClO4, CF3COONa), and stationary phase type (Zorbax Eclipse XDB, Zorbax Extend) were studied as chromatographic factors. The proposed procedure implies the combination of D-optimal experimental design, indirect modeling, and polynomial-modified Gaussian model, while grid point search method was selected for the final choice of the experimental conditions which lead to the best possible stationary phase performance for basic solutes. Good agreement between experimentally obtained chromatogram and simulated chromatogram for chosen experimental conditions (25% acetonitrile, 75 mM of NaClO4, pH 4.00 on Zorbax Eclipse XDB column) confirmed the applicability of the proposed procedure. The additional point was selected for the verification of proposed procedure ability to distinguish changes in solutes' elution order. Simulated chromatogram for 21.5% acetonitrile, 85 mM of NaClO4, pH 5.00 on Zorbax Eclipse XDB column was in line with experimental data. Furthermore, the values of left and right peak half-widths obtained from indirect modeling were used in order to evaluate performances of differently modified stationary phases applying a half-width plots approach. The results from half-width plot approach as well as from the proposed procedure indicate higher efficiency and better separation performance of the stationary phase extra densely bonded and double end-capped with trimethylsilyl group than the stationary phase with the combination of end-capping and bidentate silane bonding for chromatographic analysis of basic solutes in RP-HPLC systems with chaotropic agents. Graphical abstract ᅟ.

Influence of the mobile phase and molecular structure parameters on the retention behavior of protonated basic solutes in chaotropic chromatography.

In this study, we present novel insights into the pH-dependent retention behavior of protonated basic solutes in chaotropic chromatography. To this end, two sets of experiments were performed to distinguish between mobile phase pH and ionic strength effects. In the first set, the ionic strength (I) was varied with the concentration of NaPF6 and additives that adjusted the mobile phase pH, while in the second set, I was kept constant by adding the appropriate amount of NaCl. In each set, the retention behavior of 13 analytes was qualitatively examined in 21 chromatographic systems, which were defined by the NaPF6 concentration in their aqueous phases (1-50mM) and the pH of their mobile phases (2, 3 or 4); the acetonitrile content was fixed at 40%. The addition of NaCl significantly reduced the differences among retention factors at studied pH values due to the effect of the Na+ ions on PF6-adsorption to the stationary phase and the magnitude of the consequential development of the surface potential. A quantitative description of the observed phenomenon was obtained by an extended thermodynamic approach. The contribution of ion-pair formation in the stationary phase to the retention of the solutes was confirmed across models at the studied pH values in the set with varying I. In the systems with a constant I, the shielding effect of the Na+ ions on the surface charge lowered the attractive surface potential and diminished the aforementioned interactions and hence the effect of the mobile phase pH on analyte retention. Eventually, we developed a readily interpretable empirical retention model that simultaneously takes into account analyte molecular structures and the most relevant chromatographic factors. Its coefficients have clear physical meaning, and owing to its good predictive capabilities, the model could be successfully used to clarify the contributions of analyte molecular structures and chromatographic factors to the specific processes underlying separation in chaotropic chromatography.

Investigation into the phenomena affecting the retention behavior of basic analytes in chaotropic chromatography: Joint effects of the most relevant chromatographic factors and analytes' molecular properties.

The aim of this study was to systematically investigate the phenomena affecting the retention behavior of structurally diverse basic drugs in ion-interaction chromatographic systems with chaotropic additives. To this end, the influence of three factors was studied: pH value of the aqueous phase, concentration of sodium hexafluorophosphate, and content of acetonitrile in the mobile phase. Mobile phase pH was found to affect the thermodynamic equilibria in the studied system beyond its effects on the analytes' ionization state. Specifically, increasing pH from 2 to 4 led to longer retention times, even with analytes which remain completely protonated. An explanation for this phenomenon was sought by studying the adsorption behavior of acetonitrile and chaotropic additive onto stationary phase. It was shown that the magnitude of the developed surface potential, which significantly affects retention - increases with pH, and that this can be attributed to the larger surface excess of acetonitrile. To study how analytes' structural properties influence their retention, quantitative structure-retention modeling was performed next. A support vector machine regression model was developed, relating mobile phase constituents and structural descriptors with retention data. While the ETA_EtaP_B_RC and XlogP can be considered as molecular descriptors which describe factors affecting retention in any RP-HPLC system, TDB9p and RDF45p are molecular descriptors which account for spatial arrangement of polarizable atoms and they can clearly relate to analytes' behavior on the stationary phase surface, where the electrostatic potential develops. Complementarity of analytes' structure with that of the electric double layer can be seen as a key factor influencing their retention behavior. Structural diversity of analytes and good predictive capabilities over a range of experimental conditions make the established model a useful tool in predicting retention behavior in the studied chromatographic system.

Testing the capability of a polynomial-modified gaussian model in the description and simulation of chromatographic peaks of amlodipine and its impurity in ion-interaction chromatography.

In this paper, the capability of a polynomial-modified Gaussian model to relate the peak shape of basic analytes, amlodipine, and its impurity A, with the change of chromatographic conditions was tested. For the accurate simulation of real chromatographic peaks the authors proposed the three-step procedure based on indirect modeling of peak width at 10% of peak height (W0.1), individual values of left-half width (A) and right-half width (B), number of theoretical plates (N), and tailing factor (Tf). The values of retention factors corresponding to the peak beginning (k(B)), peak apex (k(A)), peak ending (k(E)), and peak heights (H0) of the analytes were directly modeled. Then, the investigated experimental domain was divided to acquire a grid of appropriate density, which allowed the subsequent calculation of W0.1, A, B, N, and Tf. On the basis of the predicted results for Tf and N, as well as the defined criteria for the simulation the following conditions were selected: 33% acetonitrile/67% aqueous phase (55 mM perchloric acid, pH 2.2) at 40°C column temperature. Perfect agreement between predicted and experimental values was obtained confirming the ability of polynomial modified Gaussian model and three-step procedure to successfully simulate the real chromatograms in ion-interaction chromatography.