Cryostructuring of Polymeric Systems: 62 Preparation and Characterization of Alginate/Chondroitin Sulfate Cryostructurates Loaded with Antimicrobial Substances.

Targeted drug release is a significant research focus in the development of drug delivery systems and involves a biocompatible polymeric carrier and certain medicines. Cryostructuring is a suitable approach for the preparation of efficient macroporous carriers for such drug delivery systems. In the current study, the cryogenically structured carriers based on alginate/chondroitin sulfate mixtures were prepared and their physicochemical properties and their ability to absorb/release the bactericides were evaluated. The swelling parameters of the polysaccharide matrix, the amount of the tightly bound water in the polymer and the sulfur content were measured. In addition, FTIR and UV spectroscopy, optical and scanning microscopy, as well as a standard disk diffusion method for determining antibacterial activity were used. It was shown that alginate/chondroitin sulfate concentration and their ratios were significant factors influencing the swelling properties and the porosity of the resultant cryostructurates. It was demonstrated that the presence of chondroitin sulfate in the composition of a polymeric matrix slowed down the release of the aminoglycoside antibiotic gentamicin. In the case of the NH2-free bactericide, dioxidine, the release was almost independent of the presence of chondroitin sulfate. This trend was also registered for the antibacterial activity tests against the Escherichia coli bacteria, when examining the drug-loaded biopolymeric carriers.

Influence of succinylation of a wide-pore albumin cryogels on their properties, structure, biodegradability, and release dynamics of dioxidine loaded in such spongy carriers.

The goal of this study was to reveal how the chemical modification, succinylation in this case, of the wide-pore serum-albumin-based cryogels affects on their osmotic characteristics (swelling extent), biodegradability and ability to be loaded with the bactericide substance - dioxidine, as well as on its release. The cryogels were prepared via the cryogenic processing (freezing - frozen storage - thawing) of aqueous solutions containing bovine serum albumin (50 g/L), denaturant (urea or guanidine hydrochloride, 1.0 mol/L) and reductant (cysteine, 0.01 mol/L). Freezing/frozen storage temperatures were either -15, or -20, or -25 °C. After defrosting, spongy cryogels were obtained that possessed the system of interconnected gross pores, whose shape and dimensions were dependent on the freezing temperature and on the type of denaturant introduced in the feed solution. Subsequent succinylation of the resultant cryogels caused the growth of the swelling degree of the pore walls of these spongy materials, resulted in strengthening of their resistance against of trypsinolysis and gave rise to an increase in their loading capacity with respect to dioxidine. With that, the microbiological tests showed a higher bactericidal activity of the dioxidine-loaded sponges based on the succinylated albumin cryogels as compared to that of the drug-carriers based on the non-modified protein sponges.

The influence of centrifugation and inoculation time on the number, distribution, and viability of intratubular bacteria and surface biofilm in deciduous and permanent bovine dentin.

The present study aimed to assess the influence of centrifugation and inoculation time on the number, distribution, and viability of intratubular bacteria and surface monospecies E. faecalis biofilm. MATERIALS AND METHODS: Forty-four semicylindrical specimens cut from primary (n = 22) and permanent (n = 22) bovine teeth were randomly assigned to the experimental groups. Teeth of each type were inoculated with E. faecalis with and without centrifugation for 1 and 14 days. The number, localization, viability of bacteria and depth of their penetration were assessed with bacterial culturing of dentin shavings, scanning electron microscopy (SEM) and confocal laser electron microscopy (CLSM). Three-way ANOVA with post-hoc Tukey test were used to assess the influence of different experimental setups on dentin infection. RESULTS: Severe dentin infection was observed in permanent and deciduous teeth after centrifugation and 1-day incubation: bacteria reached the full length of dentinal tubules and colony-forming units were too numerous to count. The volume of green fluorescence didn't differ significantly in permanent teeth compared with deciduous (p = 1.0). After 1-day stationary inoculation, small number of cultivable bacteria and few viable bacteria in dentinal tubules were found in both groups. After 14-day stationary inoculation, the dentin infection according to CLSM was deeper in deciduous teeth compared with permanent (p = 0.006 and p = 0.019 for centrifugation and stationary inoculation, respectively). CONCLUSION: The most even and dense dentin infection was observed in primary and permanent bovine teeth after centrifugation and 1-day inoculation, and in deciduous teeth after 14-day stationary inoculation.

Short-term fluctuations of sugar beet damping-off by Pythium ultimum in relation to changes in bacterial communities after organic amendments to two soils.

Previously, oscillations in beet seedling damping-off by Pythium ultimum, measured as area under the disease progress curve (AUDPC), were demonstrated after incorporation of organic materials into organic and conventional soils. These periodic fluctuations of P. ultimum infections were cross-correlated with oscillations of copiotrophic CFU at lags of 2 to 4 days. For this article, we investigated whether bacterial communities and microbial activities fluctuated after a disturbance from incorporation of organic materials, and whether these fluctuations were linked to the short-term oscillations in AUDPC of beet seedling damping-off and bacterial populations (CFU) in soil. Soil microbial communities studied by polymerase chain reaction-DGGE analysis of 16S DNA after isolation of total DNA from soil and microbial activities measured as CO(2) emission rates were monitored daily for 14 days after addition of grass-clover (GC) or composted manure (CM) into organic versus conventional soils. Similar to our previous findings, AUDPC and density of copiotrophic bacteria oscillated with time. Fluctuations in species richness (S), Shannon diversity index (H), and individual amplicons on DGGE gels were also detected. Oscillations in AUDPC were positively cross-correlated with copiotrophic CFU in all soils. Oscillations in AUDPC were also positively cross-correlated with 19 to 35% of the high-intensity DNA fragments in soils amended with GC but only 2 to 3% of these fragments in CM-amended soils. AUDPC values were negatively cross-correlated with 13 to 17% of the amplicons with low average intensities in CM-amended soils, which were not correlated with densities of copiotrophic CFU. CO(2) emission rates had remarkable variations in the initial 7 days after either of the soil amendments but were not associated with daily changes in AUDPC. The results suggest that infection by P. ultimum is hampered by competition from culturable copiotrophic bacteria and some high-intensity DGGE amplicons, because AUDPC is cross-correlated with these variables at lags of 1 to 4 days. However, negative cross-correlations with low-intensity DNA fragments indicate that P. ultimum infection could also be suppressed by antagonistic bacteria with low densities that may be nonculturable species, especially in CM amended soil. The organic soil generally had lower AUDPC values, higher bacterial diversity, and negative cross-correlations between AUDPC and low-intensity DNA fragments (after CM amendment), indicating that specific bacteria that do not attain high densities may contribute to P. ultimum suppression in organic soils.

Daily changes of infections by Pythium ultimum after a nutrient impulse in organic versus conventional soils.

Bacterial populations (CFU) have been shown to oscillate in wavelike patterns after nutrient impulses in previous studies. The amplitudes and periods of oscillations could possibly be used as indicators of soil health analogous to the stability and resilience of biological populations widely accepted as indicators for ecosystem health. Limited plant and animal disease outbreaks can also be viewed as a manifestation of a healthy soil ecosystem. Two pot experiments were carried out to verify whether damping-off of beet seedlings by Pythium ultimum, measured as area under the disease progress curve (AUDPC), fluctuated over time after incorporation of organic materials into organic versus conventional soils, and to investigate whether daily dynamics of AUDPCs were linked to the dynamics of microbial populations and chemical parameters. AUDPCs oscillated significantly over time when Pythium bioassays were initiated daily after addition of ground grass and clover shoots (GC) into unplanted soils. Similar oscillations with significant harmonics of AUDPC were also observed in composted manure (CM)-amended soils but with smaller amplitudes than in GC-amended soils. The AUDPC harmonics in amended soils had periods similar to those of CFU of copiotrophic bacteria. Cross-correlation analysis demonstrated that periodic fluctuations of P. ultimum infections (AUDPCs) did not coincide with those of copiotrophic CFU but were shifted in phase. It appears that competition or antagonism from some fast-growing bacteria influenced pathogen infections, because these bacterial populations were growing and dying. Soil chemical variables, including pH, dissolved organic carbon, and NO(3)(-)-N, and NH(4)(+)-N contents, changed significantly in the initial 7 days after a nutrient impulse into soils. These changes were cross-correlated with copiotrophic CFU with time lags of approximately 1 to 2 days but were seldom associated with daily changes in AUDPCs. Organically managed soils always had lower AUDPC ratios of amended to nonamended treatments, indicating that organic materials showed stronger suppressive abilities to P. ultimum in organic than in conventional soils. The oscillations in AUDPCs and copiotrophic CFU in amended organic soil also had smaller amplitudes than in amended conventional soil. These results suggested that organically managed soils had a greater resistance and resilience to the disturbance of the amendments and, therefore, could be considered healthier than conventionally managed soils.

Transfer of enteric pathogens to successive habitats as part of microbial cycles.

Escherichia coli O157:H7 gfp and Salmonella enterica Typhimurium gfp passed through six successive habitats within a microbial cycle. Pathogen cultures were introduced into cow dung or fodder. Microscopically observed cells and CFUs were monitored in fodder, dung, dung-soil mix, rhizosphere and phyllosphere of cress or oat plants grown in infested dung-soil mix, and in excrements of snails or mice fed with contaminated cress or oat shoots. Both methods were sensitive enough to monitor cells and CFUs throughout the chain. There was a positive correlation between cells and CFUs. Both pathogens declined through the successive habitats, but with unexpected increased densities on plants compared to dung-soil mix. Pathogen densities were higher in the phyllosphere than the rhizosphere of cress, but for oat plants this was reverse. Survival in dung was better after passage through the digestive tract of cows than after introduction of cultures into dung. Positive correlations between pathogens and copiotrophic bacteria (CB) and dissolved organic carbon (DOC) were observed in dung and dung-soil mixtures, but at low DOC contents CB densities were higher than pathogen densities. Thus, the pathogens are able to cycle through different habitats, surviving or growing better at high DOC concentrations, but maintaining population densities that are sufficiently high to cause disease in humans.

Isolation and partial characterization of bacterial strains on low organic carbon medium from soils fertilized with different organic amendments.

A total of 720 bacterial strains were isolated from soils with four different organic amendment regimes on a low organic carbon (low-C) agar medium (10 µg C ml(-1)) traditionally used for isolation of oligotrophs. Organic amendments in combination with field history resulted in differences in dissolved organic carbon contents in these soils. There were negative correlations between total and dissolved organic carbon content and the number of isolates on low-C agar medium, whereas these correlations were absent for bacterial strains isolated from the same soil on high-C agar medium (1,000 µg C ml(-1)). Repeated transfers (up to ten times) of the isolates from low-C agar medium to fresh low- and high-C agar media were done to test for exclusive growth under oligotrophic conditions. The number of isolates exclusively growing under oligotrophic conditions dropped after each subsequent transfer from 241 after the first to 98 after the third transfer step. Identification on the basis of partial 16S rRNA gene sequences revealed that most of the 241 isolates (as well as the subset of 98 isolates) belong to widespread genera such as Streptomyces, Rhizobium, Bradyrhizobium, and Mesorhizobium, and the taxonomic composition of dominant genera changed from the first transfer step to the third. A selected subset of 17 isolates were further identified and characterized for exclusive growth on low-C agar medium. Two isolates continued to grow only on low-C agar medium up to the tenth transfer step and matched most closely with Rhizobium sullae and an uncultured bacterium on the basis of the almost full-length 16S rRNA gene. It was concluded that the vast majority of strains which are isolated on low-C agar media belong to the trophic group of microorganisms adapted to a "broad range" of carbon concentrations, including well-known and widespread bacterial genera. Oligotrophy is a physiological, not a taxonomic property, and can only be identified by cultural means so far. We showed that true oligotrophs that are unable to grow on high carbon media are rare and belong to genera that also contain broad-range and copiotrophic strains.

Influence of temperature fluctuations on Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in cow manure.

The effects of four average temperatures (7, 16, 23 and 33 degrees C) and daily oscillations with three amplitudes (0, +/-4, +/-7 degrees C) on the survival of the enteropathogens Escherichia coli O157:H7 and Salmonella serovar Typhimurium were investigated in small microcosms. Manure was inoculated with a green fluorescent protein transformed strain of either pathogen at 10(7) cells g(-1) dry weight. Samples were collected immediately after inoculation, and 1 and 2 weeks after inoculation for E. coli O157:H7, and immediately and after 2 and 3 weeks for Salmonella serovar Typhimurium. Population densities were determined by dilution plating and direct counting. In addition, total bacterial CFUs were determined. Growth and survival data were fitted to a modified logistic model. Analysis of the estimated parameter values showed that E. coli O157:H7 survived for shorter periods of time and was more sensitive to competition by the native microbial community than Salmonella serovar Typhimurium. Survival of both pathogens significantly declined with increasing mean temperatures and with increasing amplitude in daily temperature oscillations. The results indicated that responses of enteropathogens to fluctuating temperatures cannot be deduced from temperature relationships determined under constant temperatures.