Biochemical Evidence for Formate Transfer in Syntrophic Propionate-Oxidizing Cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei

The hydrogenase and formate dehydrogenase levels in Syntrophobacter fumaroxidans and Methanospirillum hungatei were studied in syntrophic propionate-oxidizing cultures and compared to the levels in axenic cultures of both organisms. Cells grown syntrophically were separated from each other by Percoll gradient centrifugation. In S. fumaroxidans both formate dehydrogenase and hydrogenase levels were highest in cells which were grown syntrophically, while the formate-H₂ lyase activities were comparable under the conditions tested. In M. hungatei the formate dehydrogenase and formate-H₂ lyase levels were highest in cells grown syntrophically, while the hydrogenase levels in syntrophically grown cells were comparable to those in cells grown on formate. Reconstituted syntrophic cultures from axenic cultures immediately resumed syntrophic growth, and the calculated growth rates of these cultures were highest for cells which were inoculated from the axenic S. fumaroxidans cultures that exhibited the highest formate dehydrogenase activities. The results suggest that formate is the preferred electron carrier in syntrophic propionate-oxidizing cocultures of S. fumaroxidans and M. hungatei.     

Bovine mammary epithelial cell cultures for the study of mammary gland functions

In the present study, the analysis of epithelial cells derived from various sources was undertaken, beginning from the mammary gland tissue through the primary cultures and their subsequent passages. The objective of the study was the comparative analysis of the stage in which the epithelial cells obtained from individuals in different lactation cycles and disparate phases of cell culture growth are the most suitable for morphological research and analysis of gene expression activity. The cultures of primary bovine mammary epithelial cells and passages were identified morphologically using immunocytochemical methods. After positive identification, real-time PCRs were performed for the analysis of the expression level of casein genes, whey protein genes, and butyrophilin gene. The most stable reference genes in real-time PCRs for the mammary gland tissue and cell cultures were also determined. Of the reference genes, the UXT and GAPDH genes appeared to be the most stable ones for the mammary gland tissue samples and epithelial cell cultures. The results obtained allowed concluding that the mammary gland samples collected from heifers constituted the most effective material for the initiation of primary cultures. The primary cultures formed characteristic for the mammary gland tissue dome structures, which images were obtained using confocal microscopy. The highest levels of expression of the CSN1S1, CSN1S2, CSN2, and CSN3 genes were detected in primary cultures. The levels of expression of whey protein genes (LALBA and BGL) were highest in the second passage. The most abundant expression of the BTN1A1 gene was observed in primary cultures and the third passage. On the basis of the whole experiment, it can be concluded that primary cultures and cells of the second passage derived from heifer individuals appeared to be the best materials for the analysis of mammary gland function and gene expression activity.     

Mechanisms of antibody formation: I. Early in vivo proliferation of mouse spleen T cells as an initial step in antibody formation

Spleen cultures prepared from mice injected 24 hr earlier with 2 × 10⁶?2 × 10⁸ sRBC and challenged in vitro with sRBC produced 10 times more anti-sRBC IgM PFC than cultures prepared from uninjected mice. The effect was specific for the particular species of foreign RBC injected in vivo. In vitro responses to TNP were also increased in spleen cultures prepared from animals injected 24 or 12 hr earlier with carrier RBC alone, directly implicating carrier-specific T cells in this process. Similar enhancements of PFC formation occurred in cultures prepared from mice which had been injected with sRBC 24 and 48 hr earlier, but which were exposed to lethal irradiation at 1 hr after injection of antigen, if their spleens were shielded extracorporeally during irradiation. This finding indicated that in vivo recruitment of antigen-reactive extrasplenic X-ray-sensitive cells from the circulating lymphocyte pool by the spleen could not account for the observed enhancement.Proliferation in the spleen of antigen-reactive T cells, commencing 12–20 hr after the administration of antigen, was demonstrated by the tritiated thymidine pulse technique. An 8-hr hot-pulse given to spleen cell cultures from normal animals at 20 hr after in vitro challenge with antigen did not affect the rate of generation of IgM-producing cells; however, administration of a similar pulse to cultures which were initiated at 12 or at 20 hr after the in vivo injection of sRBC eliminated the enhanced generation of PFC and delayed the in vitro response to sRBC by 24 hr.Spleen cell cultures were prepared from mice which had been injected in vivo with sRBC at 12, 20, and 70 hr earlier, and 8- to 10-hr hot pulses were given immediately after initiation of the cultures. The cultures were then challenged with sRBC-TNP; antibody responses to TNP were greatly reduced in hot-pulsed cultures prepared from mice injected in vivo with carrier RBC at 12 or 20 hr prior to initiation of the cultures. In contrast, antibody responses to TNP observed in hot-pulsed cultures prepared from mice which had been injected with carrier RBC at 70 hr prior to initiation of the cultures were generally similar to those of nonpulsed 70 hr control cultures. This result suggests that the onset of T helper cell proliferation begins within 12–20 hr after injection of antigen, but subsides in vivo within 70 hr. By that time, the antigen-reactive T cells have already differentiated to perform their helper function.In spite of the triggering of T-cell proliferation during the first 24 hr after injection of antigen, spleen cell cultures prepared from mice which had been injected 24 hr earlier in vivo with 2 × 10⁸ sRBC produced only minimal numbers of anti-sRBC PFC if no antigen was added to the cultures. The presence of unprocessed antigen thus appears to be a requirement for B-cell proliferation in vitro, even after T-cell division has been triggered. This finding is consistent with earlier suggestions that the function of “helper” T cells may not be limited to passive transport of antigenic determinants to B cells. Evidence is also presented to support the contention that the antigen-reactive T cell involved in this process may have to undergo cell division in order to develop “helper” capacity.     

Resistance of Salmonellae Isolated in 1959 and 1960 to Tetracyclines and Chloramphenicol

The resistance of cultures of Salmonella typhimurium to tetracyclines and chloramphenicol has been examined periodically. Although none of 200 cultures isolated prior to 1948 was resistant to the antibiotics, 5% of 100 cultures from man and 9% of 100 cultures from fowls which were isolated in 1956 and 1957 were resistant to tetracyclines. Among 158 cultures isolated from man and 100 cultures isolated from fowls in 1959 and 1960, 13.9 and 29%, respectively, were resistant to tetracyclines. In the last series, cultures resistant to chloramphenicol were found for the first time. Among 150 cultures of other Salmonella serotypes from man and 137 similar cultures isolated from fowls in 1959 and 1960, 5.3 and 8%, respectively, were found resistant to tetracyclines. There is no obvious explanation for the higher percentage of resistant strains occurring in S. typhimurium than in other serotypes.     

Distinct Effects of Abelson Kinase Mutations on Myocytes and Neurons in Dissociated Drosophila Embryonic Cultures: Mimicking of High Temperature

Abelson tyrosine kinase (Abl) is known to regulate axon guidance, muscle development, and cell-cell interaction in vivo. The Drosophila primary culture system offers advantages in exploring the cellular mechanisms mediated by Abl with utilizing various experimental manipulations. Here we demonstrate that single-embryo cultures exhibit stage-dependent characteristics of cellular differentiation and developmental progression in neurons and myocytes, as well as nerve-muscle contacts. In particular, muscle development critically depends on the stage of dissociated embryos. In wild-type (WT) cultures derived from embryos before stage 12, muscle cells remained within cell clusters and were rarely detected. Interestingly, abundant myocytes were spotted in Abl mutant cultures, exhibiting enhanced myocyte movement and fusion, as well as neuron-muscle contacts even in cultures dissociated from younger, stage 10 embryos. Notably, Abl myocytes frequently displayed well-expanded lamellipodia. Conversely, Abl neurons were characterized with fewer large veil-like lamellipodia, but instead had increased numbers of filopodia and darker nodes along neurites. These distinct phenotypes were equally evident in both homo- and hetero-zygous cultures (Abl/Abl vs. Abl/+) of different alleles (Abl¹ and Abl⁴) indicating dominant mutational effects. Strikingly, in WT cultures derived from stage 10 embryos, high temperature (HT) incubation promoted muscle migration and fusion, partially mimicking the advanced muscle development typical of Abl cultures. However, HT enhanced neuronal growth with increased numbers of enlarged lamellipodia, distinct from the characteristic Abl neuronal morphology. Intriguingly, HT incubation also promoted Abl lamellipodia expansion, with a much greater effect on nerve cells than muscle. Our results suggest that Abl is an essential regulator for myocyte and neuron development and that high-temperature incubation partially mimics the faster muscle development typical of Abl cultures. Despite the extensive alterations by Abl mutations, we observed myocyte fusion events and nerve-muscle contact formation between WT and Abl cells in mixed WT and Abl cultures derived from labeled embryos.     

Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)

The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.     

Differential fluctuation in virulence and VOC profiles among different cultures of entomopathogenic fungi

Insect-passaged cultures of entomopathogenic fungi grown on potato dextrose agar media have been shown to have altered virulence and profiles of volatile compounds. The present study demonstrated the pathogenic status of FS₀ (in vitro) and FS₁ and FS₂ (insect-passaged cultures grown on PDA) cultures of Metarhizium anisopliae (strains 406 and 02049) and Beauveria bassiana by a non-choice assay, in which filter paper was inoculated with fungal spores at a concentration of 1 × 10⁷ spores/ml. The FS₁ and FS₂ cultures of M. anisopliae strain 02049 and B. bassiana produced conidia with high virulence, and the volatile profiles of these conidia comprised relatively lower percentages of branched-alkanes than conidia from the FS₀ cultures. In contrast, the conidia from an FS₀ culture of M. anisopliae strain 406 had somewhat elevated virulence levels, but their volatile profile had <2% branched-alkanes. The FS₁ and FS₂ cultures of M. anisopliae strain 406 did not gain virulence, and these cultures showed a decline in virulence along with major alteration of their volatile profiles. Their volatile profiles mainly comprised branched-alkanes. The volatile profiles of the FS₁ and FS₂ cultures lacked n-tetradecane, which was an important component of all the virulent cultures. Four compounds, 2-phenylpropenal, 2,5,5-trimethyl-1-hexene, n-tetradecane and 2,6-dimethylheptadecane, were detected only from the virulent cultures, suggesting that low LT₅₀ values were probably due to the production of these compounds. This is the first report to characterize volatiles from FS₀, FS₁ and FS₂ cultures of entomopathogenic fungi; its utility in different aspects opens an interesting area for further investigations.     

Plasmodium berghei: The in Vitro immune response

Following primary in vitro Stimulation by Plasmodium berghei, IgM titers by the indirect fluorescent antibody test (IFAT) were negative in in vitro reconstituted syngeneic mouse spleen cultures containing T cells and macrophages, or B cells and macrophages, or macrophages alone, but IgM titers of 1:20 were obtained from cultures containing B cells, T cells, and macrophages. IFAT IgG titers were negative for cultures with T cells and macrophages together, or macrophages alone, but rose to 1:40 with cultures containing B cells and macrophages and 1,80 with cultures of B cells, T cells, and macrophages together. After a second in vitro challenge, IgM and IgG titers were similar to the stimulated cultures from immunized mice; the IgM titer reached only 1:20 but the IgG titer rose to 1:160. Total immunoglobulin was higher in the cultures from immunized mice than from in vitro primed cultures.     

Allelopathic effects of the dinophyte Prorocentrum minimum on the growth of the bacillariophyte Skeletonema costatum

We investigated growth interactions between the dinophyte Prorocentrum minimum and the bacillariophyte Skeletonema costatum using bi-algal cultures under axenic conditions. When low cell densities of P. minimum and high cell densities of S. costatum were inoculated into the same medium, growth of P. minimum was suppressed. Other inoculum combinations resulted in reduced S. costatum maximum cell densities. A mathematical model was used to simulate growth and interactions of P. minimum and S. costatum in bi-algal cultures. The model indicated that P. minimum always outcompeted S. costatum over time. Enriched filtrate from low-density P. minimum cultures significantly stimulated S. costatum growth, but enriched filtrate from high-density P. minimum cultures notably inhibited the growth of S. costatum. Growth of P. minimum was not affected by enriched filtrate from cultures of P. minimum at any density. Filtrates of P. minimum cultures were fractionated by ultrafiltration (molecular weight cutoff >3000 Da), and retentate that included polysaccharide(s) significantly inhibited the growth of S. costatum.     

Food Microorganisms Influencing the Growth of Staphylococcus aureus

Some 870 cultures of predominating micro-organisms were isolated from market samples of hamburger, fresh pork sausage, fresh fish fillets, stewing beef, frozen chicken pot pie, frozen corn, frozen peas, and pasteurized and raw milk, before and after storage at different temperatures. The isolates were screened for their ability to influence the growth of Staphylococcus aureus strain 196E by means of spot-plate tests on APT and nutrient agars at 25 C. The 438 cultures that influenced the growth of S. aureus were retested on spot plates at 15, 30, and 42 C. After elimination of replicates, the 143 remaining cultures were classified into species, genera, or groups, and 14 different cultures were tested for their influence on the growth of S. aureus in APT broth at 25 C. Over half of the effective cultures inhibited S. aureus and less than half were stimulatory. Pork sausage had the highest proportion of inhibitory cultures, and stewing beef had the lowest. APT agar was better than nutrient agar for screening, and incubation at 15 C gave more effector organisms than at 30 and 42 C. Most of the lactic acid bacteria were inhibitory, but other groups of bacteria contained more stimulatory cultures than inhibitory ones. The three Escherichia coli cultures were stimulatory, but most other Escherichia cultures were inhibitory. Aerobacter and Paracolobactrum isolates were mostly stimulatory. Cultures of other kinds of bacteria were more or less evenly distributed between inhibitory ones and stimulatory ones. Genera containing mostly inhibitory bacteria were Streptococcus, Leuconostoc, and Lactobacillus. Inhibitory species were E. freundii and E. intermedia. Tests with S. aureus in broth indicated that all cultures inhibitory according to spot plates were inhibitory in broth, but stimulation on spot plates did not always indicate the same phenomenon in broth.     

Phylogenetic and Functional Diversity Within Toluene-Degrading,Sulphate-Reducing Consortia Enriched from a Contaminated Aquifer

Three toluene-degrading microbial consortia were enriched under sulphate-reducing conditions from different zones of a benzene, toluene, ethylbenzene and xylenes (BTEX) plume of two connected contaminated aquifers. Two cultures were obtained from a weakly contaminated zone of the lower aquifer, while one culture originated from the highly contaminated upper aquifer. We hypothesised that the different habitat characteristics are reflected by distinct degrader populations. Degradation of toluene with concomitant production of sulphide was demonstrated in laboratory microcosms and the enrichment cultures were phylogenetically characterised. The benzylsuccinate synthase alpha-subunit (bssA) marker gene, encoding the enzyme initiating anaerobic toluene degradation, was targeted to characterise the catabolic diversity within the enrichment cultures. It was shown that the hydrogeochemical parameters in the different zones of the plume determined the microbial composition of the enrichment cultures. Both enrichment cultures from the weakly contaminated zone were of a very similar composition, dominated by Deltaproteobacteria with the Desulfobulbaceae (a Desulfopila-related phylotype) as key players. Two different bssA sequence types were found, which were both affiliated to genes from sulphate-reducing Deltaproteobacteria. In contrast, the enrichment culture from the highly contaminated zone was dominated by Clostridia with a Desulfosporosinus-related phylotype as presumed key player. A distinct bssA sequence type with high similarity to other recently detected sequences from clostridial toluene degraders was dominant in this culture. This work contributes to our understanding of the niche partitioning between degrader populations in distinct compartments of BTEX-contaminated aquifers.     

Selection of lactose-adapted cells in vinca minor and Dantura innoxia cultures; location and characterization of β-galactosidase and lactase activities

Lactose-adapted cells were obtained from Datura innoxia sucrose growing calli cultures and from Vinca minor glucose growing calli cultures. Lactose adaptation process points out the homogeneity of the cell population towards lactose uptake in V. minor cultures while it reveals the presence of heterogeneous population in D. innoxia cultures.In both species, lactose hydrolysis was only occurring in the cells; no lactase activity was detected in the culture medium. An intermittent lactase activity was determined in a cell-free extract during the culture period. Lactase activity was detected in Vinca glucose grown cells as well in Datura lactose-adapted cells cultured in absence of lactose; so lactase is a constitutive enzyme. Galactose liberated during lactose hydrolysis was not toxic for thecells; it was released into the culture medium and not metabolized in Vinca cultures while it was metabolized in Datura cultures at the end of the culture period.     

Quinine and quinidine production by cinchona leaf,root and unorganized cultures

Serially propagated Cinchona ledgeriana and C. succirubra (Rubiaceae) leaf, root and unorganized suspension cultures established from germinated seeds were studied for quinine and quinidine production. Leaf organ cultures were grown and subcultured in Murashige and Skoog's Revised Tobacco Medium supplemented with benzyladenine; root organ cultures were grown on the same medium supplemented with indolebutyric acid; and unorganized suspension cultures were grown on the same medium supplemented with 2,4-dichlorophenoxyacetic acid and benzyladenine. On a dry weight basis, leaf organ cultures of C. ledgeriana contained 0.06 % quinine and 0.05 % quinidine and of C. succirubra contained 0.04 % quinine and 0.04 % quinidine. No quinine and quinidine were detected in either root organ or unorganized suspension cultures.     

Rapid Differentiation Between Nocardia and Streptomyces by Paper Chromatography of Whole-Cell Hydrolysates

Whole-cell hydrolysates were prepared from 58 strains of nocardiae and streptomycetes. Strains morphologically intermediate between the two genera and morphological variants of the same strains were included. Paper chromatograms made from the whole-cell hydrolysates clearly demonstrated meso-diaminopimelic acid as a major constituent of cultures of Nocardia spp., and LL-diaminopimelic acid as a major constituent of cultures of Streptomyces spp. In cultures of ten strains of N. madurae and of three of N. pelletieri, meso-diaminopimelic acid predominated, thereby supporting the assignment of these species to the genus Nocardia.     

Performance Evaluation of the Verigene Gram-Positive and Gram-Negative Blood Culture Test for Direct Identification of Bacteria and Their Resistance Determinants from Positive Blood Cultures in Hong Kong

Background

A multicenter study was conducted to evaluate the diagnostic performance and the time to identifcation of the Verigene Blood Culture Test, the BC-GP and BC-GN assays, to identify both Gram-positive and Gram-negative bacteria and their drug resistance determinants directly from positive blood cultures collected in Hong Kong.

Methods and Results

A total of 364 blood cultures were prospectively collected from four public hospitals, in which 114 and 250 cultures yielded Gram-positive and Gram-negative bacteria, and were tested with the BC-GP and BC-GN assay respectively. The overall identification agreement for Gram-positive and Gram-negative bacteria were 89.6% and 90.5% in monomicrobial cultures and 62.5% and 53.6% in polymicrobial cultures, respectively. The sensitivities for most genus/species achieved at least 80% except Enterococcus spp. (60%), K.oxytoca (0%), K.pneumoniae (69.2%), whereas the specificities for all targets ranged from 98.9% to 100%. Of note, 50% (7/14) cultures containing K.pneumoniae that were missed by the BC-GN assay were subsequently identified as K.variicola. Approximately 5.5% (20/364) cultures contained non-target organisms, of which Aeromonas spp. accounted for 25% and are of particular concern. For drug resistance determination, the Verigene test showed 100% sensitivity for identification of MRSA, VRE and carbapenem resistant Acinetobacter, and 84.4% for ESBL-producing Enterobacteriaceae based on the positive detection of mecA, vanA, bla _OXA and bla _CTXM respectively.

Conclusion

Overall, the Verigene test provided acceptable accuracy for identification of bacteria and resistance markers with a range of turnaround time 40.5 to 99.2 h faster than conventional methods in our region.     

Transformation of Elemental Mercury by Bacteria

The fate and impact of elemental mercury in closed bacterial cultures were examined. The quantity of elemental mercury oxidized by bacteria ranged from small amounts for Pseudomonas aeruginosa, P. fluorescens, Escherichia coli, and Citrobacter to essentially all of the added elemental mercury for Bacillus subtilis and B. megaterium. The percentage of the total mercury in the system associated with bacterial cells ranged from 18.6 43.2%. Growth of the two Pseudomonas species was inhibited by elemental mercury, whereas growth of the other cultures was not distinguishable from that in mercury-free controls. No methylmercury was formed by the six cultures within 48 h.     

Effects of the Sex Ratio Organism on IN VITRO Differentiation of Drosophila Embryonic Cells

Effects of Sex Ratio Organism (SRO) on the development of embryonic cells of Drosophila melanogaster were studied in vitro. The single embryo culture technique and a histochemical staining method were employed to distinguish male embryo cultures from female ones. SRO-infected Zwⁿ females were crossed to wild-type males, and their embryos were separately dissociated and cultured in vitro. Cell growth and differentiation in each culture were examined by phase contrast microscopy and described both qualitatively and quantitatively. Neurons, imaginal disk cells and plasmatocyte-like cells barely differentiated in male (Zw ⁿ/Y) cultures, whereas muscle and fatbody cells were not so severely affected. These symptoms were generally specific to male cultures. Female (Zwⁿ/+) cultures were relatively normal. These observations are consistent with the results of mosaic analysis. The susceptibility of cells to SRO is discussed in relation to the dose of X chromosome in the cells.     

Characterization of precipitates formed by H2S-producing, Cu-resistant Firmicute isolates of Tissierella from human gut and Desulfosporosinus from mine waste

The purpose of this study was to characterize precipitates formed in anaerobic, H₂S-producing cultures of two Tissierella isolates and Desulfosporosinus strain DB. The cultures were grown in Cu-containing media as part of a larger study of Cu resistance in anaerobic sulfidogens. The Tissierella strains produced H₂S from peptone. Desulfosporosinus formed H₂S from peptone or through dissimilatory sulfate reduction with lactate. Tissierella cultures precipitated iron phosphate, vivianite, but no crystalline phases or Cu sulfides were detected. Multiple Cu sulfides, including chalcopyrite and covellite, were detected in Desulfosporosinus cultures but vivianite was not formed. Ion microprobe spectra and electron microscopic examination showed major variation in the elemental composition and morphological differences depending on incubation conditions. Extended incubation time for at least 1–2 months increased the crystallinity of the precipitates. The results highlight biogeochemical differences in sulfide and phosphate precipitates between the two major groups of Firmicutes although they may share the same habitat including the human intestinal tract.     

Isolation of natural cultures of anaerobic fungi and indigenously associated methanogens from herbivores and their bioconversion of lignocellulosic materials to methane

This study aimed to obtain natural cultures of anaerobic fungi and their indigenously associated methanogens from herbivores and investigate their ability to degrade lignocelluloses to methane. Eight natural cultures were obtained by Hungate roll tube technique. The fungi were identified as belonging to Piromyces, Anaeromyces and Neocallimastix respectively by microscopy, and the methanogens as Methanobrevibacter spp. by 16S rRNA gene sequencing. In vitro studies with rice straw showed that these cultures degraded 33.5-48.3% substrate and produced 0.33-0.84 mmol/(100 ml culture) methane. Two cultures were further selected for their ability to degrade different lignocellulosic materials and could produce 0.38-1.27 mmol/(100 ml culture) methane. When methanogens were inhibited, the lignocellulose-degrading ability of cultures significantly reduced. In conclusion, natural cultures of anaerobic fungi with indigenously associated methanogens with high fiber degradation ability were obtained, and these cultures may have the potential in industrial use in lignocelluloses degradation and methane production.