An in vitro model for investigating intestinal adhesion of potential dairy propionibacteria probiotic strains using cell line C2BBe1

AIMS: The purposes of this study were to screen the adhesion properties of dairy propionibacteria strains and evaluate whether C2BBe1 could be used in the screening of potential probiotic strains. METHODS AND RESULTS: Thirteen dairy propionibacteria strains and two control strains, Lactobacillus acidophilus MJLA1 and Bifidobacterium lactis BDBB2, were tested for adhesion to C2BBe1. Electron microscopic observations demonstrated that the control strains, L. acidophilus MJLA1 and B. lactis BDBB2, had similar adhesive ability to C2BBe1 as had been previously shown to Caco-2. Only one of the 13 strains of dairy propionibacteria, strain P. jensenii 702, demonstrated adhesion to C2BBe1. CONCLUSIONS: C2BBe1 can provide an alternative to Caco-2 for assessing in vitro adhesion properties of probiotic strains. Adhesion properties of dairy propionibacteria were strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: C2BBe1 is highly suitable for application in bacterial adhesion studies, and was used successfully to select a new potential probiotic.     

Bacteria produce the volatile hydrocarbon isoprene

Various bacterial species, both Gram-negative and Gram-positive, were found to produce the volatile hydrocarbon isoprene (2-methyl-1,3-butadiene). Out of the tested cultures, Bacillus produced the most isoprene. The production of isoprene from bacteria was confirmed by gas chromatography-mass spectrometry. Media and growth effects on isoprene production were investigated: growth in rich media led to higher levels of isoprene than growth in minimal media, and highest isoprene emission rates were seen in log-phase cultures. Temperature profiles for bacterial isoprene production showed an optimum of 45°C and were suggestive of an enzymatic mechanism for isoprene formation.     

Norepinephrine as a growth stimulating factor in bacteria--mechanistic studies

Catecholamines (norepinephrine, epinephrine, dopamine) enhance the growth of several species of gram-negative bacteria. Since catechol rings are known siderophores in bacteria, the administration of catecholamines may enhance growth by improving iron uptake in growth-limiting media, serving as auxiliary siderophores. We have tested the iron content in bacterial growth media which are known to support rapid growth and "slow growth" media. Additionally, we have examined the uptake of 3H-norepinephrine, to determine whether the catecholamine is actually taken into the bacteria or is merely adsorbed to the outside of the bacteria. Finally, we have been examining the supernatants produced by culturing bacteria with norepinephrine. These supernatants have been shown to have the capacity to enhance growth of naive cultures of bacteria, and are suggested to contain an "autoinducer of growth". We have found that both fast-growth and slow-growth media contain similar concentrations of iron, and that these levels do not change in most supernatants from NE-supplemented bacterial cultures. Examination of culture supernatants from NE-supplemented bacteria under different temperature conditions reveals some interesting differences. First, culture supernatant from NE-treated Escherichia coli, cultured at 37 degrees C, when examined by HPLC, exhibits a change in the norepinephrine content over time which is not seen in supernatant from 21 degrees C cultures or other media treatments. Second, the 37 degrees C culture NE-supplemented E. coli supernatant was significantly more effective in enhancing growth of three bacterial species than any other culture method other than NE-supplementation itself (this includes supernatant from NE-supplemented cultures of the other two species as well as supernatants from unsupplemented cultures of all three species).     

Migration of Salmonella typhi through Intestinal Epithelial Monolayers: An In Vitro Study

This study characterizes the transmigration of enteroinvasive Salmonella typhi in vitro, using a human intestinal epithelial cell line as a model of small intestinal epithelium. C2BBe cells, a subclone of CACO-2 with a highly differentiated enterocytic phenotype, were grown to maturity on Transwell filters. S. typhi Ty2 and the vaccine strain, Ty21a, the S. typhi mutant X7344 and parent strain SB130, and S. typhimurium 5771 in logarithmic phase were introduced to the upper chamber of the filter units. Numbers of bacteria in the lower chamber, TER and permeability of the monolayer to mannitol were measured over time. Monolayers were examined by light, electron and confocal microscopy to determine the pathway of bacterial transmigration, and intracellular bacteria were estimated by gentamicin assay. Epithelial cell injury was quantified by light microscopy. S. typhi transmigrated earlier and in larger numbers than S. typhimurium, inducing marked changes in electrical resistance and permeability. Unlike S. typhimurium, S. typhi selected epithelial cells in small number and caused their death and extrusion from the monolayers leaving holes through which S. typhi transmigrated. Ty2 consistently transmigrated in larger numbers and with more injury to monolayers than Ty21a. S. typhi crosses the monolayers of C2BBe cells by a paracellular route in contrast to the transcellular pathway described for other Salmonellae. This may be related to the unique pathophysiology of S. typhi infection and the restricted host specificity of this pathogen. In these assays the vaccine strain, Ty21a, is slightly less invasive than its parent, though more invasive than S. typhimurium.     

Mass Production Potential of the Bacto-Helminthic Biocontrol Complex Heterorhabditis indica - Photorhabdus luminescens

Heterorhabditis indica is a potential agent for the biological control of grubs in sugarcane fields in India. The type strain LN 2 was transferred to monoxenic cultures on its symbiont Photorhabdus luminescens and successfully produced on solid media. In liquid cultures, a mean dauer juvenile yield of 457 000 was obtained with a maximum of 648 000 per ml. Comparatively high yields have not been reported before. Therefore, costs related to the liquid culture production of H. indica will be lower than for other entomopathogenic nematodes currently used in biocontrol. Different bacterial clones had no significant influence on the dauer juvenile yields in liquid media. The exit from the dauer juvenile stage (recovery) after inoculation and the number of hermaphrodites significantly decreased when culture temperature was increased from 25-30 ° C; the dauer juvenile yields were not affected. The cell density of P. luminescens in batch cultures was higher at 25 and 30 ° C than at growth temperatures of 35 and 37 ° C. In continuous culture, the bacterial growth was inhibited when the growth temperature reached 38 ° C. After approximately 60 h, the bacteria adapted to higher temperature and the growth rate increased again. When the temperature was further increased to 40 ° C, the bacterial growth was inhibited.     

Fish flesh agar medium — a suitable experimental medium for the detection of spoilage bacteria

The spoilage characteristics of bacterial strains were studied by growing them at 28 ± 2°C in agar and broth media prepared with sterile fish and prawn flesh homogenates. The percentage of spoilers found among the bacterial isolates tested, as shown by odour production and halo zone formation, was independent of the source of flesh used. Indole and fluorescent pigment production were also observed in the broth.Pseudomonas, Vibrio andAcinetobacter exhibited faster growth in flesh media than in the usual artificial media. Decrease of protein and lipid concentration in the clear zone of agar media suggests the utilization of the available substrate by spoilage bacteria.     

Response in Soil of Cupriavidus necator and Other Copper-Resistant Bacterial Predators of Bacteria to Addition of Water, Soluble Nutrients, Various Bacterial Species, or Bacillus thuringiensis Spores and Crystals

Soil was incubated with various species of bacteria, Bacillus subtilis, or Bacillus thuringiensis spores and crystals. These were added to serve as potential prey for indigenous, copper-resistant, nonobligate bacterial predators of bacteria in the soil. Alternatively, the soil was incubated with soluble nutrients or water only to cause potential indigenous prey cells to multiply so the predator cells would multiply. All of these incubation procedures caused excessive multiplication of some gram-negative bacteria in soil. Even greater multiplication, however, often occurred for certain copper-resistant bacterial predators of bacteria that made up a part of the gram-negative response. Incubation of the soil with copper per se did not give these responses. In most cases, the copper-resistant bacteria that responded were Cupriavidus necator, bacterial predator L-2, or previously unknown bacteria that resembled them. As was the case for C. necator and L-2, these new bacteria did not use glucose, had white colonies, produced copper-related growth initiation factor (GIF), and attacked B. thuringiensis spores on laboratory media. The results were different, however, when B. thuringiensis spores and crystals per se were added to the soil. The copper-resistant bacterial response in the soil did not, to any extent, include C. necator-like bacteria. Instead, the main copper-resistant bacterial predators that developed had yellow colonies and did not resemble C. necator or L-2 in other ways. They were not seen before, and they did not develop on the addition of B. subtilis spores to soil. Apparently, they could not produce a C. necator-like GIF. Nevertheless, they did respond very quickly to B. thuringiensis spores and crystals in soil, as if a GIF of some sort were involved. These results suggest that, under various conditions of soil incubation, gram-negative bacterial predators of bacteria multiply and that several copper-resistant types among them can be detected, counted, and isolated by plating dilutions of the soil onto media containing excess copper.     

The Effect of the Cultivation Method and the Growth Phase on the Lipopolysaccharide Composition of Yersinia pseudotuberculosis

Effects of the cultivation method (suspension cultures in a liquid nutrient broth or colonies on a solid agarized medium) and the growth phase on the lipopolysaccharide (LPS) composition of Yersinia pseudotuberculosis(O : Ib serovar, strain KS 3058) grown in cold (5°C) were studied. The amount of the LPS synthesized by cells depended on the bacteria growth phase for both media. The LPS acylation degree was constant, whereas the length of the O-specific polysaccharide chain varied with the culture age and for both media achieved maximum in the stationary growth phase. The bacteria cultivation on the nutrient agar stimulated more intensive synthesis of LPS, which were extracted more easily, had longer polysaccharide O-chains, and were more toxic than LPS of the bacteria cultivated in the liquid medium. It was proposed that the cultivation of Yersinia pseudotuberculosisin cold as colonies on the agar surface increases the bacterial virulence.     

Effects of epidermal growth factor and linoleic acid on lipid contents in human intestinal C2BBe1 cells

Epidermal growth factor (EGF) was reported to regulate triacyl glycerol synthesis in various cells. Linoleic acid and its metabolites were thought to modulate the signal transduction of growth factors. This study determined whether linoleic acid regulated the effect of EGF on lipid contents in human intestinal C2BBe1 cells. Confluent cells were incubated with serum-free medium (control), EGF (45 ng/mL), linoleic acid (42 microg/mL), or combined EGF (45 ng/mL) and linoleic acid (42 microg/mL) for 48 h. The results showed EGF and linoleic acid significantly increased intracellular cholesterol and triglyceride levels compared with the control and combined groups. EGF was a more potent stimulator for triacyl glycerol synthesis in C2BBe1 cells than linoleic acid. However, intracellular cholesterol and triglyceride levels did not differ between the control and combined groups. The secretion of cholesterol and triglyceride into the medium by C2BBe1 cells did not differ among four groups. Both EGF and linoleic acid strongly stimulated the expression of EGF receptor mRNA in C2BBe1 cells at 48 h compared with the control and combined groups. Therefore, EGF and linoleic acid increased triacyl glycerol synthesis in C2BBe1 cells through stimulating the expression of EGF receptor mRNA. The effect of EGF and linoleic acid on this lipogenesis was reversed in the presence of both EGF and linoleic acid by downregulating the expression of EGF receptor mRNA.     

Cytotoxicity and Calcium-Dependent Antigen of Yersinia

The relationship between invasiveness and calcium dependency was examined in various strains of Yersinia enterocolitica and Y. pseudotuberculosis by using established cell lines. Infection with calcium-dependent bacteria resulted in the formation of microvilli and the adherence of bacteria on the cell surface, and the adherent bacteria were ingested 1.5 hr after infection. Morphological changes in the cells became visible 2 to 3 hr after infection, and intracellular multiplication of the ingested bacteria was noted. When the cells were incubated with bacteria at 37 C for 1.5 hr and then at 25 C, however, the morphological changes in the infected cells were not observed. No isogenic strains that had lost calcium dependency for growth at 37 C were able to elicit the morphological changes in the cells, though they possessed the ability to adhere to and penetrate the cells. The antigen(s) supposedly related to cytotoxicity of the calcium-dependent Yersinia was sought by using antibodies prepared against calcium-dependent bacteria and then absorbed with calcium-independent bacteria and with calcium-independent bacterial cytosol. Double diffusion tests between the antisera and bacterial cytosol extracts revealed the presence of an antigen which was a cytoplasmic substance common to all calcium-dependent but not calcium-independent strains of Y. enterocolitica and Y. pseudotuberculosis.     

BACTERIA THAT INDUCE MORPHOGENESIS IN ULVA PERTUSA (CHLOROPHYTA) GROWN UNDER AXENIC CONDITIONS1

Marine foliaceous green macroalgae such as Ulva lose their typical morphology when cultured aseptically in defined synthetic media. However, after reinfection by certain marine bacteria (isolated from unialgal cultures of Ulva pertusa Kjellman), the organisms regain their typical foliaceous or tubular morphology. To investigate the morphogenesis (MG) induced in U. pertusa by bacteria, we isolated and identified bacteria with MG activity on U. pertusa and studied the distribution of such bacteria in seawater and on various marine macroalgae. We isolated 1555 bacterial strains from 18 species of marine macroalgae (six Chlorophyta, five Phaeophyta, and seven Rhodophyta), from seawater and from sediment collected at the beach at Omaezaki, Shizuoka Prefecture; Japan. Of these, 676 bacterial strains (43.5%) showed MG activity. They were classified into six bacterial groups, Flavobacterium, Vibrio, Pseudomonas, Deleya, Escherichia, and gram-positive cocci. These bacteria were ubiquitous among the samples and were not specific to U. pertusa. Several plant growth regulators had no MG activity. Filter-sterilized supernatants of culture media of MG-active bacteria strains did not induce MG. Cocultivation of Ulva with active bacterial strains is so far the only way to induce the MG effect, which suggests that for MG direct contact between Ulva and the bacterial strain is necessary.     

Growth and disinfestation of 6 different bacteria in embryogenic suspension cultures of cotton

Summary Growth of 6 different common laboratory bacteria (Escherichia coli, Flavobacterium balustrum, Xanthomonas maltophilia, Enterobacter cloacae, Pseudomonas fluorescens, and Agrobacterium tumefaciens) in a bacterial medium, fresh plant medium, and spent plant media was initially measured. In all cases, bacteria grew best in the bacterial medium followed by the fresh plant medium. The spent plant medium did not support growth of the bacteria and apparently was actively toxic to bacterial cells. Proliferating, embryogenic suspension cultures of cotton (Gossypium hirsutum) were then inoculated with these 6 different bacteria. Two to three d following bacterial inoculation, embryogenic tissues were placed in various concentrations of bleach for various amounts of time, rinsed with sterile water, and placed on a bacterial culture medium. Clumps of embryogenic tissue which showed no visible bacterial growth after 3 d of culture were then transferred to an agar-solidified plant tissue culture medium to determine viability of bleachdisinfested tissues. Viable, single pieces of the disinfested embryogenic tissue were then used to reinitiate embryogenic suspension cultures. Treatment of contaminated tissue with a 1% bleach solution for 1–5 min resulted in the highest recovery of viable, disinfested tissues using 5 of the 6 bacteria. It was not possible to remove F. balustrum from clumps of embryogenic tissue without also killing the plant tissue.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid Salaries and research support were provided by an Ohio Academy of Sciences/National Science Foundation summer internship award to JAS and by State and Federal funds appropriated to OSU-OARDC. OARDC Journal Article No. 391-89     

Dissolved organic carbon in a humic lake: effects on bacterial production and respiration

Allochthonous matter was the main source of carbon for pelagic bacteria in a humic lake, accounting for almost 90% of the carbon required to support observed bacterial growth. The estimated contribution from zooplankton excretion was of the same magnitude as direct phytoplankton release, both accounting for 5–7% of bacterial demands for dissolved carbon. Bacteria were an important source of carbon both for heterotrophic phytoplankton and for filter feeding zooplankton species, further stressing the role of humus DOC in overall lake productivity. The high contribution of allochthonous DOC implies a stoichiometry of dissolved nutrients with a surplus of C relative to P. The high P cell quota of bacteria suggest that under such conditions they are P-limited and act like net consumers of P. Excess C will be disposed of, and bacterial respiration rate will increase following a transition from carbon-limited bacterial growth towards mineral-nutrient-limited growth. Thus the high community respiration and frequent CO₂-supersaturation in humic lakes may be caused not only by the absolute supply of organic C, but also by the stoichiometry of the dissolved nutrient pool.     

A simulation model for the effect of predation on bacteria in continuous culture

A simulation model was developed for the carbon (C), nitrogen (N), and phosphorus (P) content of bacteria and their medium in a chemostat. Cell components distinguished included the structural component, synthetic machinery, building blocks and intermediates, C reserves, ammonium (NH₄), orthophosphate (PO₄), and polyphosphate. Growth, incorporation of substrates, and production of waste products were related to physiological status, as indicated by the amounts of various cell components. The model was fitted to data from chemostats on the chemical composition of bacteria growing in C-, N-, and P-limiting media and was used to explore the consequences of predation on bacterial populations. In C-limiting media predation (without the return of nutrients to the medium by the predator) increased NH₄ uptake in spite of a decrease in bacterial biomass. In N-limiting media predation decreased both biomass and the rate of N uptake. These results were accounted for by the effect of growth rate on bacterial N demand. In C-limiting media the return of NH₄ and PO₄ by the predator did not change the effect of predation on bacteria. But in N-limiting media the return of nutrients decreased the effect of predation on biomass, and stimulated respiration and NH₄ uptake by the bacteria. The effect of growth rate on the chemical composition of bacteria was proposed as a possible explanation of the stimulatory effect of predators on bacteria.     

Growth of Chlorella vulgaris and associated bacteria in photobioreactors

The aim of this study was to test three flat plate photobioreactor configurations for growth of Chlorella vulgaris under non‐axenic conditions and to characterize and quantify associated bacterial communities. The photobioreactor cultivations were conducted using tap water‐based media to introduce background bacterial population. Growth of algae was monitored over time with three independent methods. Additionally, the quantity and quality of eukaryotes and bacteria were analysed using culture‐independent molecular tools based on denaturing gradient gel electrophoresis (PCR‐DGGE) and quantitative polymerase chain reaction (QPCR). Static mixers used in the flat plate photobioreactors did not generally enhance the growth at the low light intensities used. The maximum biomass concentration and maximum specific growth rate were 1.0 g l^?1 and 2.0 day^?1 respectively. Bacterial growth as determined by QPCR was associated with the growth of C. vulgaris. Based on PCR‐DGGE, bacteria in the cultures mainly originated from the tap water. Bacterial community profiles were diverse but reproducible in all flat plate cultures. Most prominent bacteria in the C. vulgaris cultures belonged to the class Alphaproteobacteria and especially to the genus Sphingomonas. Analysis of the diversity of non‐photosynthetic microorganisms in algal mass cultures can provide useful information on the public health aspects and unravel community interactions.     

Growth efficiencies of freshwater bacterioplankton

The growth efficiency of freshwater bacteria was examined in continuous cultures. One series of experiments was carried out using generation times from 50 to 200 hours and aged, normal, and enriched media, all of natural origin. Another series of experiments examined the bacterial growth efficiency during the growth season in eutrophic Frederiksborg Slotssø, in relation to changes in the planktonic communities and to factors controlling the bacterial incorporation of ³H-thymidine. Attachment of bacteria to the inner surfaces of the experimental flasks was examined using various types of bottles, adding glass tubes to the bottles, and measuring ³H-thymidine incorporation and direct cell counts of attached and free-living bacteria. Attachment of bacteria varied, and in one example up to 36% of the thymidine incorporation was by attached bacteria after 4 days. It was calculated that 36% of attached bacteria caused an underestimation of the growth efficiency of 11%. The mean growth efficiency tended to decrease with generation time using enriched medium (47 to 19%) and aged medium (35 to 12%), and tended to decrease with medium quality (enriched > normal > aged media) from 37% to 27%. The only significant difference in growth efficiency occurred in relation to generation time, in samples with enriched medium (unpaired t-test, P < 0.05). The overall mean value for all generation times and media was 30% (SEM = 3%, n = 24). From April to October, the growth efficiency was determined 5 times in samples from Frederiksborg Slotssø. The overall mean value was 31% (SEM = 3%, n = 30), and there was no significant change in the growth efficiency during the period measured. In June, three bioassay experiments revealed that carbon limitation controlled bacterial incorporation of ³H-thymidine, whereas additions of phosphate and nitrate did not change the incorporation rates. The narrow range of growth efficiencies obtained in this study (mean 31%, SEM = 2%, n = 54) suggests that changes in substratequality in the media applied and in the eutrophic samples examined causes only subtle changes in the growth efficiency.     

Effect of growth media and phase on Raman spectra and discrimination of mycobacteria

When developing a Raman spectral library to identify bacteria, differences between laboratory and real world conditions must be considered. For example, culturing bacteria in laboratory settings is performed under conditions for ideal bacteria growth. In contrast, culture conditions in the human body may differ and may not support optimized bacterial growth. To address these differences, researchers have studied the effect of conditions such as growth media and phase on Raman spectra. However, the majority of these studies focused on Gram‐positive or Gram‐negative bacteria. This article focuses on the influence of growth media and phase on Raman spectra and discrimination of mycobacteria, an acid‐fast genus. Results showed that spectral differences from growth phase and media can be distinguished by spectral observation and multivariate analysis. Results were comparable to those found for other types of bacteria, such as Gram‐positive and Gram‐negative. In addition, the influence of growth phase and media had a significant impact on machine learning models and their resulting classification accuracy. This study highlights the need for machine learning models and their associated spectral libraries to account for various growth parameters and stages to further the transition of Raman spectral analysis of bacteria from laboratory to clinical settings.      

Nutritional characteristics of a mixotrophic nanoflagellate,Ochromonas sp.

Autotrophic and heterotrophic growth characteristics of a nano-flagellate were investigated. The flagellate,Ochromonas sp., was isolated from the northern Baltic Sea. Autotrophic growth was poor. Axenically pregrown flagellates did not increase significantly in cell number during incubation in different inorganic media. The number of flagellates remained constant 3–5 weeks in cultures kept in the light (100mol m^–2 sec^–1), whereas in the dark, a high mortality rate was found. Uptake of inorganic¹⁴C into an acid-stable fraction indicated thatOchromonas had a functional photosynthetic apparatus. Heterotrophic growth in both liquid medium and medium containing bacteria was rapid. The maximum growth rate corresponded to a generation time of 5.3 hours. Light had no effect on heterotrophic growth. Cells pregrown onEscherichia coli minicells survived without additional bacteria as food when kept in the light, but rapid death occurred in darkness. In conclusion, heterotrophy is the major mechanism to support growth in this species ofOchromonas, but under poor environmental conditions photoautotrophy might be a strategy for survival rather than growth.     

Water and temperature relations of softrot bacteria: growth and disease development

The effect of water availability and the temperature of the growth substrate on growth and disease development of softrot bacteria were studied using artificial media and plant material. Water availability was measured as the osmotic potential of a solution (ψ_osm) and was assessed for solutions of PEG4000 and KNO₃ as artificial osmotica and for plant tissue of chicory heads. Growth of softrot bacteria was found at water potentials from ψ= -0.12 MPa to ψ= -8.0 MPa but the lag phase of the growth curve increased with decreasing water potential. The relative growth rates of the three softrot pathogens showed a sigmoidal relationship with water potential, the relative growth rates decreasing rapidly at water potentials lower than ψ= -1.5 MPa. The water potential of harvested chicory heads decreased with storage time of the harvested crop but was still within the growth limits for softrot bacteria. In relation to temperature, the relative growth rate of Erwinia carotovora subsp. carotovora (Ecc) was highest at 10°C, of Erwinia carotovora subsp. atroseptica (Eca) at 15°C and of Pseudomonas marginalis (Pm) at 5°C. Chicory heads of two cultivars, Rumba and Salsa, inoculated with Ecc, had a significantly higher disease severity at 30°C (0.72 for Rumba and 0.47 for Salsa) than at lower or higher temperatures. In conclusion, temperature and water availability during forcing of chicory were not factors limiting populations of softrot bacteria. Possibilities for crop protection thus only avail during chicory root storage. During storage a high death rate combined with a low growth rate of the softrot bacteria may result in a decrease of bacterial populations below the minimum densities needed for infection during the forcing of chicory heads.