In vivo predation and modification of the Mediterranean fruit fly Ceratitis capitata (Wiedemann) gut microbiome by the bacterial predator Bdellovibrio bacteriovorus.

AIMS: The Mediterranean fruit fly (the medfly) causes major losses of agricultural fruits. Its microbiome is mainly composed of various Enterobacteriaceae that contribute to nutrient acquisition and are associated with the fly's development. Moreover, the performance of males produced by the sterile insect technique is improved by providing mass-reared insects with specific gut bacteria. Bdellovibrio and like organisms (BALOs) are obligate predators of Gram-negative bacteria that efficiently preys upon diverse Enterobacteriaceae, making it a potential disruptor of the fly's microbiome. We hypothesized that the fly's microbiome can be targeted to control the insect. METHODS AND RESULTS: Inoculation of B. bacteriovorus as free-swimming or encapsulated cells into gut extracts significantly reduced gut bacterial abundance, sustaining predator survival. Similar treatments applied to adult flies showed that the predators also survived in the gut environment. While addition of the predators did not affect total gut bacterial abundance and end-point fly mortality, a shift in the gut community structure, measured by high-throughput community sequencing was observed. CONCLUSIONS: The bacterial predator of bacteria B. bacteriovorus can prey and survive in vivo in the medfly gut. SIGNIFICANCE AND IMPACT OF THE STUDY: This study establishes the potential of BALOs to affect the microbiome of insect hosts.

Hydrocolloid liquid-core capsules for the removal of heavy-metal cations from water.

Liquid-core capsules with a non-crosslinked alginate fluidic core surrounded by a gellan membrane were produced in a single step to investigate their ability to adsorb heavy metal cations. The liquid-core gellan-alginate capsules, produced by dropping alginate solution with magnesium cations into gellan solution, were extremely efficient at adsorbing lead cations (267 mg Pb(2+)/g dry alginate) at 25 °C and pH 5.5. However, these capsules were very weak and brittle, and an external strengthening capsule was added by using magnesium cations. The membrane was then thinned with the surfactant lecithin, producing capsules with better adsorption attributes (316 mg Pb(+2)/g dry alginate vs. 267 mg Pb(+2)/g dry alginate without lecithin), most likely due to the thinner membrane and enhanced mass transfer. The capsules' ability to adsorb other heavy-metal cations - copper (Cu(2+)), cadmium (Cd(2+)) and nickel (Ni(2+)) - was tested. Adsorption efficiencies were 219, 197 and 65 mg/g, respectively, and were correlated with the cation's affinity to alginate. Capsules with the sorbed heavy metals were regenerated by placing in a 1M nitric acid suspension for 24h. Capsules could undergo three regeneration cycles before becoming damaged.

Physical characterization of a new skin bioadhesive film.

Physical properties (roughness, gloss, mechanical, surface topography and adhesive) of a bioadhesive film for the transdermal delivery of drugs and its interactions with a skin model surface were studied. Roughness is a measurement of the small-scale variations in the height of a physical surface. No significant differences in Ra between the "x" and "y" dimensions for both the skin model and patch were detected, due to uniformity in their production. Scanning electron microscope pictures showed small particles projected from the film. Those particles resulted in increasing roughness and surface area. For the patch, gloss values measured at 20 degrees were 6.0 +/- 0.9 and at 60 degrees , 32.2 +/- 2.2 gloss units, respectively, indicating a semi-gloss material. Concerning the mechanical properties, the tensile strength of the film resulted four- to sevenfold greater than the peel force from the model skin used, indicating the suitability of the film for skin application. The adhesion to skin model depended on the amount of water used for film application and on the elapsed time between film application and removal. Finally, the model skin that was invented by Charkoudian can be used as an alternative to costly and highly variable human skin substrates since it possesses human topography.

Hydrocolloid carriers with filler inclusion for diltiazem hydrochloride release.

Hydrocolloid beads based on agarose, alginate (both 3%, w/w), or gellan (2%, w/w) were produced to study their potential as drug carriers. The beads included various fillers: talc, kaolin, calcium carbonate, potato, or corn starch (10%, w/w). After gelation, the carriers were subjected to either freeze- or vacuum-drying. The dried carriers were spheroids. The diameters of freeze- and vacuum-dried carriers ranged from 2.4 to 4.1 mm and 1.5 to 2.8 mm, respectively. The porosity values of the freeze-dried carriers were significantly higher than those of their vacuum-dried counterparts. Scanning electron microscopy (SEM) revealed that all dried carriers included internal voids that were partially occupied by the filler particles. Upon their introduction into simulated gastric fluid (3 h), followed by 6 h in intestinal fluid, all carriers were stable and underwent swelling. Release profiles of diltiazem hydrochloride from different carriers were obtained during immersion in dissolution medium. Filler inclusion (but not the type of filler) contributed to the stability of the carriers and prolonged the time of drug release (6.5-8.5 h) relative to the faster drug release from carriers that contained no filler (3.5 h). In summary, alginate, agar, and gellan beads with filler inclusion may be useful for slow drug release.

Mutual relationships between soils and biological carrier systems.

Improved viability and antagonistic activity of biocontrol agents during soil inoculation is of crucial importance to their effective application. The chitinolytic bacterium Serratia marcescens was used as a model organism to study the efficacy of freeze-dried alginate beads (in comparison to their non-dried counterparts) as possible carriers for immobilized biocontrol agents. The release of bacteria and chitinolytic enzyme from alginate beads, before and during their application in soil, was examined, and the beads' physical properties characterized. Dispersal of the alginate bead-entrapped S. marcescens in the soil resulted in high soil cell densities throughout the 35 days of the experiment. Chitin inclusion in the beads resulted in significantly higher chitinolytic activity of S. marcescens, increased dry-bead porosity and decreased stiffness. Rehydration of the dried beads (after immersion in soil) resulted in a sixfold increase in weight due to water absorption. No significant differences were found in bacterial count inside the non-dried (gel) versus dried beads. However, higher cell densities and chitinase activity were detected in soil containing dried beads with chitin than in that containing their non-dried counterparts. The biological performance of S. marcescens was examined in the greenhouse: a free cell suspension reduced bean (Phaseolus vulgaris L.) disease by 10%, while immobilized bacteria found in the dried, chitin-containing beads reduced disease by 60%.

Weight loading young chicks inhibits bone elongation and promotes growth plate ossification and vascularization.

The mechanical stimuli resulting from weight loading play an important role in mature bone remodeling. However, the effect of weight loading on the developmental process in young bones is less well understood. In this work, chicks were loaded with bags weighing 10% of their body weight during their rapid growth phase. The increased load reduced the length and diameter of the long bones. The average width of the bag-loaded group's growth plates was 75 +/- 4% that of the controls, and the plates showed increased mineralization. Northern blot analysis, in situ hybridization, and longitudinal cell counting of mechanically loaded growth plates showed narrowed expression zones of collagen types II and X compared with controls, with no differences between the relative proportions of those areas. An increase in osteopontin (OPN) expression with loading was most pronounced at the bone-cartilage interface. This extended expression overlapped with tartarate-resistant acid phosphatase staining and with the front of the mineralized matrix in the chondro-osseous junction. Moreover, weight loading enhanced the penetration of blood vessels into the growth plates and enhanced the gene expression of the matrix metalloproteinases MMP9 and MMP13 in those growth plates. On the basis of these results, we speculate that the mechanical strain on the chondrocytes in the growth plate causes overexpression of OPN, MMP9, and MMP13. The MMPs enable penetration of the blood vessels, which carry osteoclasts and osteoblasts. OPN recruits the osteoclasts to the cartilage-bone border, thus accelerating cartilage resorption in this zone and subsequent ossification which, in turn, contributes to the observed phenotype of narrower growth plate and shorter bones.

Unexpected distribution of immobilized microorganisms within alginate beads.

Immobilization refers to the prevention of free cell movement by natural or artificial means. It has always been assumed that immediately after an immobilization procedure is performed, cells are distributed homogeneously in the beads that entrap them. However, in this study, Escherichia coli and Trichoderma asperellum distribution in alginate-gel beads was found to be nonhomogeneous. In fact, there was a greater presence of cells on the surface of the alginate beads than in their cores.

Short-duration low-direct-current electrical field treatment is a practical tool for considerably reducing counts of gram-negative bacteria entrapped in gel beads.

Application of a direct-current electrical field for very short times can serve as a practical nonthermal procedure to reduce or modify the microbial distribution in gel beads. The viability of Escherichia coli and Serratia marcescens entrapped in alginate and agarose beads decreases as the field intensity and duration of electrical field increase.

Structure of dried cellular alginate matrix containing fillers provides extra protection for microorganisms against UVC radiation.

Soil microorganisms in general and biocontrol agents in particular are very sensitive to UV light. The packaging of biocontrol microorganisms into cellular solids has been developed as a means of reducing loss caused by exposure to environmental UV radiation. The bacterial and fungal biocontrol agents Pantoea agglomerans and Trichoderma harzianum were immobilized in freeze-dried alginate beads containing fillers and subjected to 254 nm UV radiation (UVC). Immobilization of cells in freeze-dried alginate-glycerol beads resulted in greater survival after UV irradiation than for a free cell suspension. Adding chitin, bentonite or kaolin as fillers to the alginate-glycerol formulation significantly increased bacterial survival. Immobilization in alginate-glycerol-kaolin beads resulted in the highest levels of survival. The transmissive properties of the dried hydrocolloid cellular solid had a major influence on the amount of protection by the cell carrier. Dried alginate matrix (control) transmitted an average of 7.2% of the radiation. Filler incorporation into the matrix significantly reduced UV transmission: Alginate with kaolin, bentonite and chitin transmitted an average of 0.15, 0.38 and 3.4% of the radiation, respectively. In addition, the filler inclusion had a considerable effect on the bead's average wall thickness, resulting in a approximately 1.5- to threefold increase relative to beads based solely on alginate. These results suggest that the degree of protection of entrapped microorganisms against UVC radiation is determined by the UV-transmission properties of the dried matrix and the cellular solid's structure. It is concluded that for maximum protection against UV-radiation-induced cell loss, biocontrol microorganisms should be immobilized in alginate-glycerol beads containing kaolin.

Direct current electrical field effects on intact plant organs.

Intact plant tissues (of hypocotyls, radicles, cotyledons and leaves) were contracted by applying a low DC electrical field through them. Stomatal opening as a result of the electrical treatment of leaves was observed, presumably due to the differential influence of the electrical treatment on guard cell turgor pressure versus turgor pressure of the surrounding epidermal cells. In addition, leakage of minerals from the treated leaves was detected (higher contents of potassium, sodium, calcium and sulfur), as was leakage of betanin from electrically treated red beet roots (higher OD value of the immersion solution with increasing time of applied electrical field). Application of such a treatment can be used for initial drying or as part of another more drastic drying method. The advantages of this method lie in its nonthermal character and its potential to increase the quality of processed foods by maintaining their "like-fresh" quality, e.g., fruits and vegetables that are less damaged by browning. An understanding of the mechanism involved in this nonthermal application can help in controlling the process and predicting its outcome.

Nitrate removal in aquariums by immobilized pseudomonas.

Biological denitrification of nitrate to nitrogen gas was examined in a freshwater and a marine aquarium. Nitrate removal in the aquarium water was accomplished with denitrifiers immobilized in a freeze-dried, alginate-starch matrix. Starch served as a bacterial carbon source and cellular matrix-strengthening filler. Freeze-dried beads were placed in canisters through which nitrate-rich aquarium water was recirculated. The freshwater aquarium (100 L) contained goldfish (Carassius auratus) at a total biomass of 390 g, whereas cichlids (Oreochromis mossambicus) were kept at a similar stocking density in the marine aquarium. Denitrification resulted in low ambient nitrate concentrations in both aquariums. The specific nitrate removal rate of the freshwater beads was significantly higher (50 microg of NO(3)-N/bead/day) than that of seawater beads (5 microg of NO(3)-N/bead/day). Differences in ambient nitrate concentrations between both aquariums and diffusion limitation of nitrate to the active denitrification sites within the beads might explain these observed differences.

Preservation of chitinolytic Pantoae agglomerans in a viable form by cellular dried alginate-based carriers.

Improved viability of Gram-negative bacteria during freeze-dehydration, storage, and soil inoculation is of crucial importance to their efficient application. The chitinolytic Pantoae (Enterobacter) agglomerans strain IC1270, a potential biocontrol agent of soil-borne plant-pathogenic fungi, was used as a model organism to study the efficacy of freeze-dried alginate-based beads (macrocapsules) as possible carriers for immobilized Gram-negative bacterial cells. These macrocapsules were produced by freeze-dehydration of alginate gel spherical beads, in which different amounts of bacteria, glycerol, and colloidal chitin were entrapped. Subsequent drying produced different unexpected structures, pore-size distributions, and changes in the outer and inner appearance of the resultant dried cellular solid. With increasing glycerol content, the proportion of larger pores increased. These structures can be related to changes in the slow-release properties of the dried beads. The amount of glycerol in the beads differed from that in the alginate solution as a result of leakage during the beads' preparation and dehydration. Entrapping 10(9) cells per bead produced from alginate solution containing 30% glycerol and 1% chitin resulted in improved (in comparison to other studies) survival prospects (95%) during freeze-drying. Moreover, immobilization of the bacterium sharply improved its survival in nonsterile irrigated and dry soils compared to bacteria in a water suspension. The results suggest that optimized conservation of Gram-negative bacteria in dry glycerol-containing alginate-based cellular solids is not only possible but applicable for a variety of uses.

Physicochemical properties and structural changes in vegetative tissues as affected by a direct current electrical field.

Cylindrical pieces of potato, sweet potato, kohlrabi, radish, and pear were interposed between a pair of electrodes, and a direct current was applied. A special custom-made apparatus enabled the use of differently shaped electrodes. The electrical field was applied for 1 min at 40 V/cm and caused a reduction in specimen weight by a minimal value of 2.7% of initial weight in sweet potato to a maximum 38.4% in pear. The affected area of the tissue resembled the shape of the electrode. Pores were produced in the tissue (from the anode side), possibly promoting slow release of minerals and other cell components from the contracted specimens. From the cathode side, cell "sealing" could be observed. Weight loss was dependent on the mechanical properties of the nontreated vegetative tissue specimens. After confirmation that all samples pass through induced electrical shrinkage, further work, executed only on potato, demonstrated that after electrical treatment the samples were less brown (higher L values). In addition, a dependence of weight loss on current intensity, electrode diameter, and surface ratio between the electrode and specimen was shown. The reduction in weight loss could be useful for initial drying of vegetative materials. Indirect proof of a decrease in enzyme activity as a result of electrical field application could be beneficial in replacing traditional methods for browning prevention.

Estimating the frequency of high microbial counts in commercial food products using various distribution functions.

Industrial microbial count records usually form an irregular fluctuating time series. If the series is truly random or weakly autocorrelated, the fluctuations can be considered as the outcome of the interplay of numerous factors that promote or inhibit growth. These factors usually balance each other, although not perfectly, hence, the random fluctuations. If conditions are unchanged, then at least in principle the probability that they will produce a coherent effect, i.e., an unusually high (or low) count of a given magnitude, can be calculated from the count distribution. This theory was tested with miscellaneous industrial records (e.g., standard plate count, coliforms, yeasts) of various food products, including a dairy-based snack, frozen foods, and raw milk, using the normal, log normal, Laplace, log Laplace, Weibull, extreme value, beta, and log beta distribution functions. Comparing predicted frequencies of counts exceeding selected levels with those actually observed in fresh data assessed their efficacy. No single distribution was found to be inherently or consistently superior. It is, therefore, suggested that, when the probability of an excessive count is estimated, several distribution functions be used simultaneously and a conservative value be used as the measure of the risk.

Enumeration and factors influencing the relative abundance of a denitrifier, Pseudomonas sp. JR12, entrapped in alginate beads.

The relative abundance of the denitrifier, Pseudomonas sp. JR12, was examined in an alginate-based entrapment complex under non-sterile, denitrifying conditions. Immuno-labeling of the Pseudomonas inoculant followed by flow cytometry (FCM) was used for determination of the relative abundance of this bacterium under the various incubation conditions. Additional information on the relative abundance of the inoculant was obtained by a quantitative enzyme-linked immunosorbent assay (ELISA) and results obtained by FCM and ELISA were compared. Ambient nitrate levels controlled the successful, long-term proliferation of the inoculant. At low ambient nitrate levels, Pseudomonas sp. remained the dominant microorganism during incubation. Higher ambient nitrate concentrations, attained by either decreasing the inoculum size of Pseudomonas sp. or raising inlet nitrate concentrations of the medium supplied to the incubation vessels, resulted in a gradual shift toward other, nitrite-accumulating denitrifiers. Thus far, most studies on the use of entrapped microorganisms for bioremediation purposes have been conducted under controlled laboratory conditions. Based on this study, conducted under non-sterile laboratory conditions, it is concluded that in-situ bioremediation using entrapped target microorganisms is bound to fail without a proper understanding of the factors that cause the target microorganism to out-compete undesired microbial invaders. Furthermore, based on the close agreement between the two detection methods used, it is concluded that flow cytometry provides a rapid and accurate tool for the detection of the relative abundance of immuno-labeled target organisms in heterogeneous microbial populations.

Analysis of the fluctuating microbial counts in commercial raw milk--a case study.

Microbial counts of raw milk from eight commercial dairy farms recorded over a period of about 2 years were obtained from a processing plant. All the counts formed irregular fluctuating series whose statistical properties were examined. In all eight cases the autocorrelation function indicated that the counts were random and had no underlying periodicity. This suggested that the fluctuations, at least to some extent, reflected the combined effect of numerous factors, some unknown, and that these factors operate randomly and independently. In five out of the eight cases the fluctuating pattern of the record could be described by a probabilistic model based on the assumption that the counts have a lognormal distribution. The validity of the model was confirmed by comparing the calculated frequencies of counts exceeding preselected levels with those actually observed in fresh data. The model enabled ranking the five sources on the basis of anticipated frequencies of excessive counts rather than on the basis of mean performance. Two sequences had clearly identified regions with different statistical characteristics. They were interpreted as a reflection of changes in the sanitary conditions at the corresponding farms. In one case, both the overall magnitudes and the fluctuation amplitude of the counts progressively decreased, a situation for which the described model as formulated was inapplicable.

Hydrocolloid coating of Xenopus laevis embryos.

A novel technology for coating single cells and embryos with thin hydrocolloid (water-soluble polymer) films has been invented and patented. Coating is different from entrapment and immobilization in that the coating around the cell is thinner, comprising only a small fraction of the cell or embryo's diameter. Xenopus laevis embryos were coated with thin films of low-methoxy pectin (LMP), alginate, and iota- and kappa-carrageenans. These gums have different compositions and structures and as such created different coatings around the fertilized cells. All coated embryos appeared to develop normally, similar to noncoated embryos. Elemental detection by ICP-AES spectroscopy revealed that the embryo can control the diffusion of excess ions to which it is exposed during the coating process. The coatings delayed hatching by 18-24 h. Consequently, at hatch the embryos were at a more developed stage than their noncoated counterparts. The hydrocolloid coating reduced the thickness of the natural jelly coating (JC). With the iota-carrageenan coating, percent hatch was maximal, while with LMP it was minimal, as a result of the films' mechanical properties and thicknesses. LMP and alginate created smoother coatings than the carrageenans. Potential interactions between the coating and the natural JC are hypothesized. Overall, coatings appear to be a suitable tool for laboratories interested in performing longer-term experiments with embryos.

Alginate coating of Xenopus laevis embryos.

Xenopus laevis eggs were coated, immediately after squeeze-stripping and fertilization, with a thin layer ( approximately 50 microm) of film based on one of three different types of alginates which varied in their mannuronic/guluronic acid ratio. The alginate was cross-linked with either Ca or Ba ions at three different concentrations. The developmental, survival, and hatching of these embryos and the swelling of their natural jelly coats or hydrocolloid coatings were studied over 7 days, while embryos were maintained in flowing aerated water at a ratio of 85 mL per embryo or at a very diminished ratio of 1.6 mL of sterile or nonsterile MMR solution per embryo. All experiments were conducted in triplicate at 20+/-1 degrees C. Oxygen was monitored continuously. Mineral content was determined in the alginate-jelly coat and within the embryos over time. The coating conferred major advantages when the ratio between the embryos and the surrounding medium was at a minimum under nonsterile conditions, perhaps as a result of the film's resistance to diffusion. In the studied systems, the coating seemed to postpone embryo hatching to a more developed stage. In addition, the coating served as a barrier to microbial contamination and thus improved survival prospects.

Oxidation of encapsulated oil in tailor-made cellular solid.

A cellular alginate solid containing oil was prepared by freeze-drying. The oil was incorporated in the matrix by emulsification in the pre-gel state. The alginate-oil gels were immersed in 60 degrees Brix sucrose solution for various periods, before freeze-drying. The extent of the collapse expressing the reduction in sample volume was affected by immersion duration and freeze-drying conditions. Sucrose diffusion during immersion followed an exponential pattern. Effective diffusivity calculated using nonlinear regression gave a value of 3.64 x 10(-)(10) m(2)/s. The effect of relative humidity on water content calculated on a dry basis excluding sucrose showed a significant increase in water content at 75% RH. Image analysis was utilized to quantify the area of the encapsulated oil droplets. The area of the droplets was divided into four subregions defined as (0.02-0.1) x 10(-)(12), (0. 1-1.0) x 10(-)(12), (1-10) x 10(-)(12), and (10-100) x 10(-)(12) m(2). A distribution resembling a Gaussian bell distribution with a maximum of 54% for the (1-10) x 10(-)(12) m(2) area range was found. The number of oil droplets was almost constant for the first three area regions, and then decreased markedly. Oxidation index was not effected by porosity at 0 and 22% RH. A 75% RH and porosity above a critical value of ca. 0.45 was found to increase oxidation significantly. Samples immersed for less than 5.5 h in sucrose solution were mechanically stronger after equilibration at 0 and 22% RH when compared to their counterpart equilibrated at 75% RH. Immersion for more than 24 h resulted in similar mechanical strength irrespective of the RH.

The behavior of hydrocolloid coatings on vegetative materials

Coating vegetative materials by gelling agents is a process characterized by four different time scales. After wetting and penetration of the vegetative skin by the gum solution, adhesion of the viscous solution to the outer layer (skin) of the coated material is possible. The gelled film (coating) collapses during further drying and adheres to the vegetative tissue. Critical surface tensions of the solid object to be coated, its apparent and real roughness, wettability of the surfaces by the gum solution, the composition and polarity of the films designed to coat the solid, and the surface tension of gum coating solutions are among the critical properties that need to be explored and changed for a successful coating process. The critical surface tensions of garlic peel and gellan and alginate films (coatings) were evaluated by Zisman plots. Garlic skin has a low surface tension compared with those of synthetic films such as polystyrene and polyethylene. A spreading technique was used to determine the surface tension of the dry film and the solid garlic skin. Surface tension was divided into dispersive and polar components. The similarity between the coating solution and the object to be coated in values of dispersive and polar components influences the spreadability of the coating gum solutions. Better compatibility between the coated object and the coating films can be achieved by incorporating surface-active agents within the coating gum solution. From the compatibility requirements detailed above, it can be concluded that tailor-made hydrocolloid coatings for different vegetative materials can only be achieved by further exploring the chemical and physical properties of the coating solutions and the coated objects.