Living cells of Staphylococcus aureus immobilized onto the capillary surface in electrochromatography: a tool for screening of biofilms

Microorganisms attach to nonliving surfaces in many natural, industrial, and medical environments, enveloped within extracellular polymeric substances. The result is a biofilm. Biofilms are reported to exist in 65-80% of bacterial infections refractory to host defenses and antibiotics therapy and are regarded as a central problem in present-day medical microbiology. Understanding of the parameters governing the interaction of antimicrobials with biofilms is thus of great interest in any attempt to increase biocide efficacy. In this work, study was made of the feasibility of using open tubular capillary electrochromatography (CEC) in bacterial biofilm studies with living cells. Staphylococcus aureus was selected as model bacterium. First, S. aureus was shown, under various conditions, to form a biofilm on the inner wall of a fused-silica capillary coated with poly(L-lysine). Optimal conditions for biofilm formation, such as bacterial concentration, growing time, and the stability of the ensemble, were preliminarily defined with conventional 96-microtiter well plates. Continuous flushing of the capillary with fresh cells meant that no growth medium was needed. The presence of biofilm in the capillary was confirmed by atomic force microscopy. Interactions between S. aureus biofilms and different antibiomicrobial agents were studied by capillary electrochromatography. The effect of five antibiotics (penicillin G, oxacillin, fusidic acid, rifampicin, vancomycin) on biofilms was examined in terms of retention factors and reduced mobilities of the antibiotics. The antibiotic susceptibility profile for S. aureus is similar as the result of minimal inhibitory concentrations registered on the 96-microtiter well plates for both planktonic and biofilm cells. The results show, for the first time, that bacterial biofilms can be studied by CEC. The technique allows highly efficient and easy characterization of interactions between S. aureus biofilms and potentially active antimicrobial compounds under different conditions. Reagent and cell consumption are minimal.     

Physical, chemical and microbiological water characteristics associated with the occurrence of Legionella in cooling tower systems

The occurrence of Legionella in water of 30 cooling tower systems was studied, as well as the relationship of its occurrence with the physical, chemical and microbiological characteristics of the waters. The samples were concentrated by membrane filtration and one part of the concentrate was acid washed. Three types of culture media, MWY, CCVC and BCYEα were used. Fourteen (47%) of the cooling systems were Legionella-positive. Numbers of Legionella in the positive systems varied between 50 and 490,000 cfu/l. Legionella pneumophila was the dominant isolate, and the serogroups 6 and 1 were the commonest ones. There was no statistically significant difference in mean water temperature between the Legionella-positive (27°C) and -negative (24°C) systems. The total number of bacteria (AODC) was lower in the Legionella-positive than in the negative systems. Also the nutrient concentrations were generally lower in the Legionella-positive cooling systems. The results suggest that different factors regulate the occurrence of Legionella than that of overall bacterial populations in the cooling tower systems.     

Three different approaches for the clarification of the interactions between lipoproteins and chondroitin-6-sulfate

Two different experimental approaches were used for obtaining a comprehensive view and understanding of the interactions between apolipoprotein B-100 (ApoB-100) of low-density lipoprotein and apolipoprotein E (ApoE) of high-density lipoprotein and chondroitin-6-sulfate (C6S) of arterial proteoglycan. The techniques employed were partial filling affinity capillary electrophoresis (PF-ACE) and continuous flow quartz crystal microbalance (QCM). In addition, molecular dynamic (MD) simulations were used to provide a supportive visual insight into the interaction mechanism. A new tool for analysis of QCM-data was utilized, i.e., adsorption energy distribution calculations, which allowed a deeper understanding of the interactions, especially at different temperatures. The PF-ACE technique probed mainly the strong adsorption interactions whereas in the MD calculations short- and long-range interactions could be distinguished. Although there are differences in the techniques, a pretty good agreement was achieved between the three approaches for the interaction of 19 amino acid peptide of ApoB with C6S giving log affinity constants of 4.66 by QCM, 5.02 by PF-ACE, and 7.39 by MD, and for 15 amino acid peptide of ApoE with C6S 5.34 by QCM, 5.28 by PT-ACE, and 4.60 by MD at physiological temperature 37.0 °C.     

Merging wildlife community ecology with animal behavioral ecology for a better urban landscape planning

Cities are extremely human-modified environments, with few existing original habitats. Local, regional and global studies have indicated scale-dependent patterns of communities in relation to urbanization. In general, species with high abundance in urban environments are generalist species, whereas specialists have declined. However, these results do not indicate directly if urban habitats are either sink or source habitats for wildlife. Reproductive success, mortality, and dispersion are key factors to improve our understanding of how to support more diverse animal communities in urban environments. We need more research on the factors affecting the behavioral responses to urbanization of species with different life-histories. Some studies have demonstrated that urbanization has clear impacts on the behavior of wildlife species, a character that is strongly related to the success of species in a given habitat. Indeed, animals can adapt to urban ecosystems behaviorally, for example, by adjusting their food preferences, foraging behavior, anti-predator behavior, or extending the length of their reproduction season. Merging community and behavioral ecology will enable a more effective conservation of remnant semi-natural habitats in urbanized landscapes.     

PFGE genotyping and antimicrobial susceptibility of Campylobacter in retail poultry meat in Estonia

In the present study, the Campylobacter isolates from retail poultry meat in Estonia were sero- and genotyped, and the antimicrobial susceptibility was determined. Forty-eight chicken (36 Estonian, 12 imported) and 22 turkey (imported) Campylobacter isolates from 580 raw broiler chicken (396 Estonian, 184 imported) and 30 turkey (imported) meat samples were studied. Of the isolates, 64 were C. jejuni, 4 C. coli, and 2 Campylobacter spp. Penner serotyping of 54 C. jejuni isolates revealed 11 different serotypes, and 22% of the isolates were nontypeable by the commercial antisera. The most common serotypes O:1,44; O:21, and O:55 accounted for 28%, 13%, and 13% of the isolates, respectively. Differences in serotype distribution were seen for chicken and turkey isolates. Genotypic characterization of all Campylobacter isolates (n=70) was performed by pulsed-field gel electrophoresis (PFGE). SmaI and KpnI yielded 29 and 34 PFGE types, respectively, revealing high diversity among isolates. The serotype distribution did not show an association with the origin of the sample, but the majority of the isolates sharing a similar PFGE genotype originated from one country. High levels of resistance to ciprofloxacin (66%), nalidixic acid (66%), tetracycline (44%), ampicillin (34%), and erythromycin (14%) were detected among the 70 Campylobacter isolates. The simultaneous resistance to two or three antimicrobial agents occurred in 60% of the isolates. The Campylobacter isolates from turkey meat had higher resistance to ampicillin, ciprofloxacin, nalidixic acid, and tetracycline than those from chicken meat. None of the chicken isolates were resistant to gentamicin, and no turkey isolates to erythromycin or gentamicin.     

Mining tailings as raw materials for reaction-sintered aluminosilicate ceramics: Effect of mineralogical composition on microstructure and properties

This paper presents studies on the utilization of aluminosilicate-based mining tailings as raw materials for mullite-based ceramics. Based on the 3:2 stoichiometric composition, mullite was synthesised by reactive sintering with a series of powder mixtures with alumina additions. X-ray diffractometry and scanning electron microscopy analyses revealed that, at the specific mineralogical composition, mullite structure formed surrounded by an amorphous glass phase in reaction-sintered powder mixtures. Results demonstrated that the chemical and mineralogical composition of mining tailings do have an effect on mullite formation possibilities and, only with the particular mineralogical composition, the mullite formation is possible regardless of the correct Al:Si ratio in tailings. Physical and mechanical properties of the formed ceramics were defined, showing comparable values to 3:2 mullite reference. Mullite structure formation enables a better thermal resistance up to above 1450?°C of the formed tailings-based ceramics compared to other aluminosilicates, reflecting their utilization potential for refractory ceramic applications.     

Modulator design for comprehensive two-dimensional gas chromatography: quantitative analysis of polyaromatic hydrocarbons and polychlorinated biphenyls

A novel cryogenic modulator was constructed for comprehensive two-dimensional gas chromatography (GC x GC). The modulator is based on two-step cryogenic trapping with CO2 and thermal desorption with electric heating. The GC x GC system included a nonpolar first-dimension column and two semipolar second-dimension columns, one connected to a flame ionization detector and the other one to a electron capture detector. A Matlab-based program, which allowed determination of peak heights and volumes, was written for the data analysis. The GC x GC system was applied for the analysis of polyaromatic hydrocarbons and polychlorinated biphenyls. The functioning of the modulator and the quantitativity of the method were studied with both peak volumes and peak heights from a three-dimensional plot. The separate peak areas from the modulated chromatogram were calculated as a comparison. The quantitative results were compared with those obtained with the same system but without the thermal modulation. The method was found to be repeatable and linear with use of peak volumes as well as peak heights. There was also good agreement with the results obtained by integration of separate peak areas. The developed GC x GC method was applied to the analysis of a Soxhlet extract of a certified sediment sample. The results were compared with the certified values.     

Determination and comparison of molar mass distributions of substituted polystyrenes and block copolymers by using thermal field-flow fractionation, size exclusion chromatography and light scattering

Anionic polymerisations of p-methoxystyrene, p-methylstyrene, p-chlorostyrene and p-cyanostyrene were carried out in tetrahydrofuran (THF) and methylcyclohexane. The block copolymers poly(styrene-b-p-methoxystyrene-b-styrene), poly(styrene-b-p-methylstyrene-b-styrene) and poly(styrene-b-p-cyanostyrene) were also polymerised by using THF as solvent. The initiators included n-butyllithium (n-BuLi) and sodium naphthalenide. The use of both n-BuLi and sodium naphthalenide usually produced higher values of _n than were calculated. In the former case this could be explained by the lower reactivity of the initiator, which has been found to aggregate in THF. The molar masses were determined by thermal field-flow fractionation (ThFFF), size exclusion chromatography (s.e.c.) and light scattering (LS). ThFFF and the s.e.c. were used for determining the molar mass distributions. The agreement between the three methods was satisfactory. © 1997 Elsevier Science Ltd.     

Evaluation of the lactose Tergitol-7, m-Endo LES, Colilert 18, Readycult Coliforms 100, Water-Check-100, 3M Petrifilm EC and DryCult Coliform test methods for detection of total coliforms and Escherichia coli in water samples

In this study we compared the reference membrane filtration (MF) lactose Tergitol-7 (LTTC) method ISO 9308-1:2000 with the MF m-Endo LES method SFS 3016:2001, the defined substrate chromogenic/fluorogenic Colilert 18, Readycult Coliforms and Water Check methods, and ready-made culture media, 3M Petrifilm EC and DryCult Coli methods for the detection of coliforms and Escherichia coli in various water samples. When the results of E. coli detection were compared between test methods, the highest agreement (both tests negative or positive) with the LTTC method was calculated for the m-Endo LES method (83.6%), followed by Colilert 18 (82.7%), Water-Check (81.8%) and Readycult (78.4%), whereas Petrifilm EC (70.6%) and DryCult Coli (68.9%) showed the weakest agreement. The m-Endo LES method was the only method showing no statistical difference in E. coli counts compared with the LTTC method, whereas the Colilert 18 and Readycult methods gave significantly higher counts for E. coli than the LTTC method. In general, those tests based on the analysis of a 1-ml sample (Petrifilm EC and DryCult Coli) showed weak sensitivity (39.5-52.5%) but high specificity (90.9-78.8%).     

Open tubular capillary electrochromatography: technique for oxidation and interaction studies on human low-density lipoproteins

A novel, open tubular capillary electrochromatographic method was developed for the in vitro oxidation of low-density lipoprotein (LDL) particles. Low-density lipoprotein particles with molar mass of approximately 2.5 MDa yielded a stable stationary phase at temperatures 25 and 37 degrees C and at pH values from 3.2 to 7.4. The quality of the coatings was not influenced by variations in the LDL concentration in the coating solutions (within the range of 2-0.015 mg/mL) with the coating procedure used in the study. Radiolabeled LDL stationary phases and scanning electron microscopy, employed to shed light on the location and coating density of LDL particles on the inner surface of the capillary wall, confirmed the presence of an LDL monolayer and almost 100% coating efficiency (99 +/- 8%). In addition, the radioactivity measurements allowed estimation of the amount of LDL present in a single capillary coating. Capillaries coated with human LDL particles were submitted to different oxidative conditions by changing the concentration of the oxidant (CuSO4), oxidation time, pH value, and temperature. The oxidation procedure was followed with electroosmotic flow mobility, which served as an indicator of the increase in total negative charges of LDL coatings, and by asymmetrical field flow fractionation, which measured the changes in size of the lipoprotein particles. The results indicated that oxidation of LDL was progressing with increasing time, temperature, and concentration of the oxidant as expected. The oxidation process was faster around neutral pH values (pH 6.5-7.4) and inhibited at acidic pH values (pH 5.5 and lower).