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Background: Burkholderia cepacia complex (Bcc) organisms produce a wide variety of potential virulence factors, including exopolysaccharides (EPS), and exhibit intrinsic resistance towards many antibiotics. In the present study we investigated the contribution of Bcc biofilm matrix components, including extracellular DNA, cepacian and poly-β-1,6-N-acetylglucosamine, to tobramycin susceptibility., Methods: The in vitro bactericidal activity of tobramycin in combination with recombinant human DNase (rhDNase), NaClO and dispersin B was tested against Bcc biofilms., Results: EPS degradation by NaClO pretreatment and specific PNAG degradation by dispersin B significantly increased the bactericidal effect of tobramycin towards some of the Bcc biofilms tested, including the strains of Burkholderia cenocepacia, B. cepacia and Burkholderia metallica. The presence of rhDNase during biofilm treatment and/or development had no influence on tobramycin activity., Conclusion: These results suggest that EPS play a role in tobramycin susceptibility of Bcc biofilms and that matrix degrading combination therapy could improve treatment of Bcc biofilm infections., (© 2013.)

Polyethylene (PE) and polypropylene (PP) were functionalized at their surfaces with cyclodextrins (CDs) in order to prevent the adhesion and proliferation of Candida albicans on medical devices made from these polymers. The surface functionalization involved the grafting of glycidyl methacrylate (GMA) after oxidative gamma-ray pre-irradiation, followed by the attachment of beta-CD and HP-beta-CD to PE-g-GMA and PP-g-GMA surfaces. The yield of CD functionalization directly depended on the amount of GMA grafted. The presence of CDs on the surface of the polymers did not compromise their cell compatibility, but remarkably changed their protein adsorption profile. In contrast to unmodified PE and PP that adsorb significant amounts of fibrinogen ( approximately 0.047 mg cm(-2)) but not albumin, the CD-modified polyethers promoted the adsorption of albumin (between 0.015 and 0.155 mg cm(-2)) and reduced the adsorption of fibrinogen. Furthermore, functionalization with CDs provided PE and PP with the capability to incorporate the anti-fungal drug miconazole (up to 0.27 mg cm(-2)), leading to reduced biofilm formation by C. albicans in an in vitro biofilm model system. Overall, the results of the work indicate that the novel approach for functionalization of PE and PP is potentially useful to reduce the likelihood of foreign body-related infections., (Copyright 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

As part of studies on the spread of infections, risk factors and prevention, several typing methods were developed to investigate the epidemiology of Aspergillus fumigatus. In the present study, 52 clinical isolates of A. fumigatus from 12 airway specimens from patients with invasive aspergillosis (hospitalized in three different centres) were characterized by short tandem repeat (STR) typing and multilocus sequence typing (MLST). These isolates were previously typed by random amplified polymorphic DNA (RAPD), sequence-specific DNA polymorphism (SSDP), microsatellite polymorphism (MSP) and multilocus enzyme electrophoresis (MLEE). STR typing identified 30 genotypes and, for most patients, all isolates were grouped in one cluster of the unweighted pair group method with arithmetic mean dendrogram. Using MLST, 16 genotypes were identified among 50 isolates, while two isolates appeared untypeable. RAPD, MSP, SSDP and MLEE allowed identification of eight, 14, nine and eight genotypes, respectively. Combining the results of these methods led to the delineation of 25 genotypes and a similar clustering pattern as with STR typing. In general, STR typing led to similar results to the previous combination of RAPD, SSDP, MSP and MLEE, but had a higher resolution, whereas MLST was less discriminatory and resulted in a totally different clustering pattern. Therefore, this study suggests the use of STR typing for research concerning the local epidemiology of A. fumigatus, which requires a high discriminatory power.

The trailing endpoint phenotype observed during testing of Candida albicans susceptibility to azoles according to the CLSI procedure is defined as a difference in MIC depending on whether the result is obtained after 24 or 48 h. This study investigated whether intrinsic differences between the EUCAST and CLSI methods could explain trailing growth. The glucose concentration in the medium and the shape of the microtitre plate wells were both found to be involved. In order to reduce the incidence of trailing growth according to the CLSI procedure, the use of higher glucose concentrations and flat-bottomed microtitre plates could be valuable improvements.

Increased resistance to fluconazole has been reported in oral, oesophageal and urinary Candida isolates, but this has not been observed commonly in genital tract isolates. The rate of isolation of Candida spp. and their susceptibility to amphotericin B, flucytosine and azoles were determined in a number of clinical practices in the city of Ghent, Belgium. Patients with symptomatic vulvovaginal candidiasis (VVC) were treated with fluconazole, and the mycological and clinical outcomes were evaluated. Isolates were identified as Candida albicans (78.6%), Candida guilliermondii (17.3%), Candida glabrata (2.6%) and Candida dubliniensis (1.3%). The rates of mycological and clinical cures were 79.5% and 100%, respectively. Women with recurrent VVC were infected more frequently by non-albicans Candida spp., but no association was found between the use of antifungal agents and the presence of non-albicans spp. In-vitro resistance to fluconazole was not detected, even among subsequent Candida isolates from nine patients for whom mycological cure was not achieved.

A flow-through enzyme immunoassay has been developed for the screening of green coffee bean samples for ochratoxin A (OA) and was later used in a survey on OA in green coffee from different countries. The test has a sensitivity of 8 ng/g, and calculated recoveries ranged from 70 to 89% and from 86 to 95% for spiked and naturally contaminated samples, respectively. There were no significant differences in within-day and between-day assay performance (P > 0.05). Green coffee samples (15 Arabica and 7 Robusta) received from an international coffee trader were analyzed for intrinsic fungal contamination, screened for OA, and subsequently confirmed by high-performance liquid chromatography (HPLC). All 22 samples were contaminated by fungal species of the genus Aspergillus, while Penicillium species were isolated from a mere 13.6% of the total number of samples. Isolates were tested for their ability to produce OA, and only 3.9% were positive. There was no correlation between occurrence of OA-producing isolates and levels of OA in contaminated samples. Results of the screening procedure showed that 4 of the 22 samples were contaminated with 8 ng/g or higher. The HPLC method confirmed that the OA levels ranged from 27 to 168 ng/g. A fifth sample, which was shown to be negative during screening, had an OA concentration of 4 ng/g. There were no false negatives or positives recorded, and the flow-through enzyme immunoassay results correlated with those obtained by HPLC.

The tetracycline galactosidase (TG) test, a new method for the detection of tetracycline residues in raw milk based on the inhibition of beta-galactosidase biosynthesis in Escherichia coli, was previously validated with spiked milk samples. It has now been applied to milk from cows treated with oxytetracycline. In view of the occurrence of false positives, related to highly elevated somatic cell counts (>10(6)/ml), the improved TG test was developed, in which a heating step (80 degrees C, 15 min) preceded the original TG test protocol. A good agreement with other assays (Delvotest SP, the Bacillus cereus microtiter test, the LacTek tetracycline milk screening test, the Charm HVS-8100 tetracycline test) as well as with high-pressure liquid chromatography was obtained. No false negatives were observed with reference to the established maximum residue limit for tetracyclines of 100 microg/kg milk.

In aquaculture, alpha-tocopheryl acetate (alpha-TA) is the main source of vitamin E used to fortify fish feed. alpha-TA in fish is often determined indirectly, i.e., by alkaline hydrolysis, followed by quantitation of "total alpha-tocopherol" (alpha-T) and subtraction of the natively present alpha-T. The aim of this study was to develop an HPLC method for the simultaneous quantitative determination of alpha-TA and free tocopherols in aquatic organisms and fish feed. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed-phase chromatography with consecutive UV and fluorescence detection of alpha-TA and tocopherols, respectively. The peak of the internal standard tocol in the fluorescence trace was used for quantitation. Linearity was achieved over the range of 0.2 to 4.2 micrograms alpha-TA per ml extract of Artemia nauplii, which would correspond to 30.7 to 614.4 micrograms/g dry mass. The within-run coefficient of variation was 1.9% at a level of 310 micrograms/g dry mass. The recovery of alpha-TA ranged from 97.7 to 100.8% (concentration = 2.1 and 20.5 micrograms/ml, n = 6). The detection limit was about 7 ng and the quantification limit on spiked samples was 0.2 microgram/ml. This method was routinely applied to determine alpha-TA and alpha-, gamma- and delta-tocopherol (alpha-T, gamma-T, delta-T) simultaneously in Artemia, fish feed, shrimp eggs and various other aquatic organisms.

A liquid chromatographic (HPLC) method has been developed for the quantitative determination of different forms of vitamin E (alpha-, gamma-, and delta-tocopherol) in aquatic organisms. The assay consists of a simple extraction with methanol containing butylhydroxytoluene (BHT) as an antioxidant, followed by reversed-phase chromatography with fluorescence detection. The efficiency of the extraction method was equivalent or superior to that of more complex approaches for the isolation of tocopherols. Linearity has been achieved over the range of 0.02 to 3 micrograms/ml for alpha-tocopherol and within-run and between-run coefficients of variation at three levels were 0.7-2.9 and 1.2-3.7%, respectively. The recovery at three concentrations ranged from 73.8 to 96.6% and the minimal quantity that could be detected was 0.6 ng. Comparable figures were obtained for gamma- and delta-tocopherol. This method has been routinely applied to determine vitamin E in Artemia, rotifers, turbot and sea bass larvae, and shrimp postlarvae.

As part of ongoing studies in the biosynthesis of the lantibiotic nisin, we investigated the proteolytic cleavage of a synthetic Fmoc-labeled decapeptide mimicking a key amino acid sequence of the precursor prenisin in extracts of a Lactococcus lactis subsp. lactis strain. Reverse-phase high-performance liquid chromatography with photodiode array detection was used to trace and purify potential enzymatic conversion products. Of the three newly appearing chromatographic peaks, one was identified by means of electrospray mass spectrometry, amino acid analysis, and amino acid sequencing as an Fmoc-labeled hexapeptide derived from cleavage at the Arg-1-Ile+1 bond. This assay will be useful to monitor the purification of the endoproteinase that reportedly cleaves prenisin at the same Arg-1-Ile+1 site present in the model substrate.

A liquid chromatographic method for the determination of oxytetracycline in bovine plasma was developed. Contrary to most current tetracycline assays involving solid-phase extraction, oxytetracycline and the internal standard tetracycline were isolated from buffered plasma by double-phase extraction with ethyl acetate-isopropyl alcohol. The latter component was found to be essential to ensure reproducible partitioning of both tetracyclines into the organic layer. Chromatography was conducted on a Lichrosorb RP8 column, compounds were eluted with 0.01 M oxalic acid-methanol-acetonitrile, and detection was at 357 nm. Linearity was observed over the 0-8.5-micrograms/mL range, and the detection limit approximated 5 ng/mL. The extraction yield was 80.4 +/- 3.4% (n = 12), and the coefficients of variation for repetitive within-run and between-run analyses at two concentration levels (0.25 and 2 micrograms/mL) were 1.0-4.3 and 2.4-7.2%, respectively. The method was applied to a pharmacokinetic study of oxytetracycline in three nonlactating cows after a single intramuscular administration of a long-acting preparation.

Quality control of albumin solutions for human use is of utmost importance because the presence of impurities can provoke adverse reactions in treated patients. Three different techniques were used to detect the presence of albumin oligomers and polymers as well as foreign proteins in commercial solutions. The relative concentrations of the former two were estimated using size-exclusion high-performance liquid chromatography. Isoelectric focusing and two-dimensional immunoelectrophoresis allowed detection of other contaminants of proteinaceous nature. The application of this combination of techniques supersedes the traditional approaches (gel filtration on polydextran gels, electrophoresis) in specificity and speed. Analysis of 34 lots of commercial albumin solutions from 22 manufacturers revealed the superior quality of preparations of placental rather than plasmatic origin.

Different high performance liquid chromatographic systems were applied to the investigation of vitamin A metabolism in subjects undergoing haemodialysis. Plasma levels of retinol, retinyl esters and retinoic acid were measured. There was a significant elevation of plasma retinol and dialysis failed to normalise this level. No correlation with plasma concentrations of creatinine or urea was found. No differences in retinyl ester and retinoic acid levels were observed between healthy subjects and haemodialysis patients. These results suggest that retinol accumulation is not caused by a deficiency in its oxidative metabolism.

A review is presented of current gas and liquid chromatographic methods for the determination of the fat-soluble vitamins A, D, E and K and the provitamin A beta-carotene in biological samples of human origin. For each vitamin, the discussion successively focuses on procedures for sample preparation, gas and liquid chromatographic systems and principles of detection. The emphasis is on liquid chromatography, which is gradually becoming a standard technique in fat-soluble vitamin assays. New trends in the liquid chromatography of these compounds include the use of smaller particles and shorter columns, to improve speed, and the advance of electrochemical detection as an alternative to absorbance and fluorescence detection. Bonded phases, both normal and reversed phase, tend to be preferred over underivatized silica as column supports. Gas chromatography remains of particular value in combination with mass spectrometry, a technique which may form the basis of reference methods. In general, despite the availability of well established analytical methods for fat-soluble vitamins, the wealth of recent literature in this area indicates that there continues to be a need for new assays with enhanced speed, specificity and sensitivity.

A rapid, specific, and sensitive liquid chromatographic assay for minocycline in human serum is described. The drug and an internal standard (oxytetracycline) were extracted into ethyl acetate from 0.5 ml of buffered serum (pH 6.5). Chromatographic separation was achieved on a 10-micrometer Lichrospher 100 CH 8 column with acetonitrile--citric acid (0.1 M) as the eluent. The column effluent was monitored at 352 nm. The assay was linear up to 3 micrograms/ml with a mean coefficient of variation of 3.3% (n = 6). An extraction recovery of 89.4 +/- 3.2% (mean +/- SD, n = 17) was obtained over the 0.5--2.6 micrograms/ml range. The detection limit averaged 50 ng/ml. A serum concentration-time profile in humans after oral intake is presented.

A rapid, sensitive and specific liquid chromatographic method is described for the determination of doxycycline in human tissues. The procedure involves mechanical homogenization of tissue samples in hydrochloric acid followed by extraction of the drug and an internal standard into ethyl acetate. Chromatographic separation is performed on a reversed phase column and allows quantitation of tissue levels as low as 0.68 nmol/g using a 200-400 mg sample. Application of the assay to tissue samples obtained from 36 patients confirmed the excellent penetration of doxycycline in organs. The method supersedes the classical microbiological assays in specificity and speed.

A reversed-phase high-performance liquid chromatographic method for quantitative doxycycline determination in human serum and urine is described. The drug was extracted from buffered (pH 6.1) serum or urine into ethyl acetate. A structural analog, demeclocycline, was added as the internal standard. A 10-cm X 2-mm i.d., 5-micrometers Lichrosorb RP8 column with acetonitrile-0.1 M citric acid as the eluent was used. The effluent was monitored at 350 nm. The extraction recovery from spiked serum was 87-8 +/- 4.3% (mean +/- SD, n = 11); for urine, a value of 92.2 +/-2.0% (mean +/- SD, n = 10) was found. Within-run and within-day relative standard deviations averaged (x = 2.5 micrograms/ml, n = 10) and 4.75% (x = 2.6 micrograms/ml, n = 9), respectively. The detection limit was estimated at 50 ng/ml of serum. No significant extra peaks were observed in chromatograms obtained on serum or urine extracts, suggesting the probable absence of metabolic processes in vivo.

A liquid chromatographic procedure for the quantitative determination of the thiazine dye azure B, the principal constituent of Romanowsky stains, is presented. Unlike previous methods relying on peak area normalization, the present approach involves real quantitation through calibration with the reference standard azure B tetrafluoroborate. The method has been used for the quality control of commercial azure B preparations and to study their stability in stock and staining solutions, either or not in the presence of eosin Y. Results suggest that highly pure azure B perchlorate meets the requirements of a reference material, useful for standardization of Romanowsky-Giemsa staining in haematology.

Chromatographic procedures have been developed for determination of the stabilizers N-acetyl-DL-tryptophan and octanoic acid in human albumin solutions. N-Acetyl-DL-tryptophan and the internal standard, N-formyl-DL-tryptophan, were separated by liquid chromatography on a reversed-phase column with UV detection at 280 nm. Deproteinization and extraction were carried out with methanol. The extraction recovery at the level of 4.9 mM was 92.5 +/- 2.5% (S.D.) (n = 10), and the average coefficient of variation (C.V.) for replicate analyses of albumin solutions (mean = 2.57, 10.44 and 17.10 mM) was 1.10% (n = 27). Octanoic acid was determined gas chromatographically as its methyl ester, with nonanoic acid as the internal standard. The sample pretreatment included acidification, extraction with hexane and derivatization with methanol-sulphuric acid. The relative recovery from albumin solutions was 89.7 +/- 5.8% (S.D.) (n = 6), and replicate determinations of the compound yielded a C.V. of 5.5% (mean = 14.82 mM, n = 9).

Several conflicting observations in the literature raised considerable doubt about the metabolic fate of doxycycline, which, like other tetracyclines, has been claimed to be metabolically inert. A double liquid chromatographic approach was used in an attempt to demonstrate the polar metabolites and/or conjugates in excreta of human volunteers who ingested the drug. Both ion-exchange and reversed-phase chromatography failed to reveal significant by-products in urine and feces, except for minor amounts of 4-epidoxycycline. In addition, enzymatic hydrolysis procedures did not present any evidence of the conjugates. Thus, the different excretion behavior of doxycycline, compared to other analogs, cannot be explained in terms of increased metabolism.

In order to study the biological fate of all-trans- and cis-canthaxanthin in the brine shrimp Artemia, a quantitative method was developed for the determination of both carotenoids and their metabolic precursors in encysted embryos (cysts), nauplii, whole animals, organs, and subcellular fractions. This method is based on nonaqueous reversed-phase chromatography, two new exhaustive extraction procedures, and the determination of proteins in the extracted residue. Hydration of Artemia cysts caused a reversible conversion of part of the all-trans- to cis-canthaxanthin. During further pre-emergence embryonic development, there was little change in the levels of both isomers. After hatching of cysts, cis-canthaxanthin was progressively isomerized to the all-trans form, while the total (all-trans + cis) canthaxanthin to protein ratio tended to remain constant. Cis-canthaxanthin rapidly became undetectable in animals fed on algae and reappeared in females at an advanced stage of the reproductive cycle. All-trans-canthaxanthin remained present during the whole Artemia life cycle in addition to its metabolic precursors echinenone and beta-carotene. The carotenoid distribution in organs and subcellular fractions indicated high affinity of cis-canthaxanthin for the female reproductive system, oocytes in general, and yolk in particular. A role for cis-canthaxanthin is suggested at an early developmental stage, i.e., in cysts, before hatching.