Non-supplemented Medium Research Articles

Diplodia seriata Isolated from Declining Olive Trees in Salento (Apulia, Italy): Pathogenicity Trials Give a Glimpse That It Is More Virulent to Drought-Stressed Olive Trees and in a Warmth-Conditioned Environment.

The fungi Botryosphaeriaceae are involved in olive declines in both the world hemispheres and in all continents where this species is cultivated. In Salento (Apulia, Italy), the Botryosphaeriaceae Neofusicoccum mediterraneum and N. stellenboschiana have been reported as the agents of a branch and twig dieback that overlaps with olive quick decline syndrome caused by Xylella fastidiosa subsp. pauca. In this study, we report the finding of Diplodia seriata, another Botryosphaeriaceae species, in Salento in Xylella fastidiosa-infected olive trees affected by symptoms of branch and twig dieback. Given that its presence was also reported in olive in the Americas and in Europe (Croatia) with different degrees of virulence, we were prompted to assess its role in the Apulian decline. We identified representative isolates based on morphological features and a multilocus phylogeny. In vitro tests showed that the optimum growth temperature of the isolates is around 25-30 °C, and that they are highly thermo-tolerant. In pathogenicity trials conducted over eleven months, D. seriata expressed a very low virulence. Nonetheless, when we imposed severe water stress before the inoculation, D. seriata significatively necrotized bark and wood in a time frame of 35 days. Moreover, the symptoms which resulted were much more severe in the trial performed in summer compared with that in autumn. In osmolyte-supplemented media with a water potential from -1 to -3 Mpa, the isolates increased or maintained their growth rate compared with non-supplemented media, and they also grew, albeit to a lesser extent, on media with a water potential as low as -7 Mpa. This suggests that olives with a low water potential, namely those subjected to drought, may offer a suitable environment for the fungus' development. The analysis of the meteorological parameters, temperatures and rainfall, in Salento in the timeframe 1989-2023, showed that this area is subjected to a progressive increase of temperature and drought during the summer. Thus, overall, D. seriata has to be considered a contributor to the manifestation of branch and twig dieback of olive in Salento. Coherently with the spiral decline concept of trees, our results suggest that heat and drought act as predisposing/inciting factors facilitating D. seriata as a contributor. The fact that several adverse factors, biotic and abiotic, are simultaneously burdening olive trees in Salento offers a cue to discuss the possible complex nature of the olive decline in Salento.

A Combination of Heavy Metals and Intracellular Pathway Modulators Induces Alzheimer Disease-like Pathologies in Organotypic Brain Slices.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by amyloid-beta (Aβ) plaques and tau neurofibrillary tangles (NFT). Modelling aspects of AD is challenging due to its complex multifactorial etiology and pathology. The present study aims to establish a cost-effective and rapid method to model the two primary pathologies in organotypic brain slices. Coronal hippocampal brain slices (150 µm) were generated from postnatal (day 8-10) C57BL6 wild-type mice and cultured for 9 weeks. Collagen hydrogels containing either an empty load or a mixture of human Aβ42 and P301S aggregated tau were applied to the slices. The media was further supplemented with various intracellular pathway modulators or heavy metals to augment the appearance of Aβ plaques and tau NFTs, as assessed by immunohistochemistry. Immunoreactivity for Aβ and tau was significantly increased in the ventral areas in slices with a mixture of human Aβ42 and P301S aggregated tau compared to slices with empty hydrogels. Aβ plaque- and tau NFT-like pathologies could be induced independently in slices. Heavy metals (aluminum, lead, cadmium) potently augmented Aβ plaque-like pathology, which developed intracellularly prior to cell death. Intracellular pathway modulators (scopolamine, wortmannin, MHY1485) significantly boosted tau NFT-like pathologies. A combination of nanomolar concentrations of scopolamine, wortmannin, MHY1485, lead, and cadmium in the media strongly increased Aβ plaque- and tau NFT-like immunoreactivity in ventral areas compared to the slices with non-supplemented media. The results highlight that we could harness the potential of the collagen hydrogel-based spreading of human Aβ42 and P301S aggregated tau, along with pharmacological manipulation, to produce pathologies relevant to AD. The results offer a novel ex vivo organotypic slice model to investigate AD pathologies with potential applications for screening drugs or therapies in the future.

In vitro maturation of African catfish (Clarias gariepinus, Burchell, 1822) oocytes results in viable larvae

In this study, we describe a protocol for in vitro maturation and ovulation of African catfish (Clarias gariepinus) postvitellogenic ovarian follicles, resulting in fertilizable and developmentally competent eggs. Such culture systems enable detailed studies of fish oocyte development and present a useful tool in assisted reproduction of fish. The maturation-inducing effects of 17α,20β-dihydroxy-4-pregnen-3-one (DHP), human chorionic gonadotropin (hCG) and recombinant human insulin-like growth factor 1 (rhIGF-1) were evaluated based on their ability to promote ooplasm translucency and germinal vesicle breakdown (GVBD), which indicates meiosis resumption. Among the tested groups, only follicles exposed to DHP underwent GVBD in a time-dependent manner, with the highest rates (>80%) observed within 10 to 12 h of incubation. Furthermore, adding rhIGF-1 or hCG prior to or during incubations with DHP did not influence the GVBD outcome. This suggested that the oocytes acquired maturational competence prior to isolation, as high concentrations of DHP (1 μg/ml) were alone sufficient to induce complete nuclear and cytoplasmic maturation; however, only 4 ± 3% of these oocytes ovulated in culture. To promote ovulation, we incorporated prostaglandins (PGs) as an additional incubation step following DHP-induced maturation. Exposure to both prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2) led to increased rupture of the follicular layer, most notably in groups with 5 μg/ml PGF2α (71 ± 8%). In addition to identifying optimal hormonal stimulation, effects of culture media pH in combination with media supplementation—in the form of bovine serum albumin (BSA) and fetal bovine serum (FBS)—were considered. Compared to results in media with pH of 7.5, significantly less follicles matured and ovulated in a more alkaline environment (pH 8.5), regardless of supplement type and concentration. Although the presence of FBS (5–20%) slightly improved ovulation rates, this benefit was not reflected in the fertilization outcomes. In vitro matured and ovulated oocytes obtained in non-supplemented media (pH 7.5) maintained their developmental competence and were successfully fertilized. The hatching rate was 39%, after which the survival rate of larvae was 8% at 72 h post fertilization (hpf).

Insight into the Mechanism of Interactions between the LL-37 Peptide and Model Membranes of Legionella gormanii Bacteria.

Legionella gormanii is a fastidious, Gram-negative bacterium known to be the etiological agent of atypical community-acquired pneumonia. The human cathelicidin LL-37 exhibits a dose-dependent bactericidal effect on L. gormanii. The LL-37 peptide at the concentration of 10 µM causes the bacteria to become viable but not cultured. The antibacterial activity of the peptide is attributed to its effective binding to the bacterial membrane, as demonstrated by the fluorescence lifetime imaging microscopy. In this study, to mimic the L. gormanii membranes and their response to the antimicrobial peptide, Langmuir monolayers were used with the addition of the LL-37 peptide to the subphase of the Langmuir trough to represent the extracellular fluid. The properties of the model membranes (Langmuir monolayers) formed by phospholipids (PL) isolated from the L. gormanii bacteria cultured on the non-supplemented (PL-choline) and choline-supplemented (PL+choline) medium were determined, along with the effect of the LL-37 peptide on the intermolecular interactions, packing, and ordering under the monolayer compression. Penetration tests at the constant surface pressure were carried out to investigate the mechanism of the LL-37 peptide action on the model membranes. The peptide binds to the anionic bacterial membranes preferentially, due to its positive charge. Upon binding, the LL-37 peptide can penetrate into the hydrophobic tails of phospholipids, destabilizing membrane integrity. The above process can entail membrane disruption and ultimately cell death. The ability to evoke such a great membrane destabilization is dependent on the share of electrostatic, hydrogen bonding and Lifshitz-van der Waals LL-37-PL interactions. Thus, the LL-37 peptide action depends on the changes in the lipid membrane composition caused by the utilization of exogenous choline by the L. gormanii.

Effects of crocin supplementation during in vitro culture of intact and half-destroyed four-cell mouse embryos.

We evaluated the effects of crocin supplementation during culture of intact and half-destroyed four-cell mouse embryos. Outcomes measured included rate of cleavage arrest, blastocyst formation, and blastocyst cell number. We used laser to create two zonal holes without blastomere destruction in Groups 1 (n=100) and 2 (n=100), and to destroy two of the four blastomeres in Groups 3 (n=150) and 4 (n=150). Embryos were cultured in groups of ten in drops of medium without (Groups 1 and 3) or with 20 μg/ml of crocin supplementation (Groups 2 and 4). Embryos in Groups 1 and 2 had no difference in the rate of cleavage arrest (6.0% vs. 7.0%, respectively; p=0.774) or blastocyst formation (89.0% vs. 86.0%, respectively; p=0.521). Neither was there a difference in the number of cells in the blastocysts (99.6±23.5 vs. 95.6± 8.2, respectively, p=0.83). Half-destroyed embryos cultured in crocin-supplemented medium (Group 4) had a lower rate of cleavage arrest (14.7% vs. 30.0%, p=0.001), and a higher rate of blastocyst formation (51.3% vs. 37.3%, p=0.015), than those in non-supplemented medium (Group 3). In blastocysts derived from half-destroyed embryos, there was no difference in the number of cells in ICM (14.5±3.9 vs. 13.7±2.9, p=0.285), TE (45.2±12.3 vs. 46.0±13.3, p=0.764), or total cells (59.7±12.2 vs. 59.7±14.8, respectively, p=0.990) among the two groups. Crocin supplementation during in vitro development of impaired embryos improved their development, but had no effect on intact embryos.

Effects of age and pasture type on the concentration and prevalence of tetracycline and macrolide resistant Enterococcus species in beef cow-calf production system

Enterococci are a normal flora of the gastrointestinal tracts of humans and animals. Enterococci can also cause life-threatening nosocomial infections. Antimicrobial-resistant Enterococcus species have been reported in the feedlot and dairy cattle productions and in meat and milk products, suggesting their foodborne importance. Cow-calf operations represent a significant segment in the beef production system by producing weaned calves. Weaned calves are brought into the feedlot to be finished for meat, and culled cows are also slaughtered for beef, primarily for ground beef products. Infection dynamics in the cow-calf operation can contribute to meat contamination. This study evaluated the effects of age and wheat grazing on the concentration and prevalence of a macrolide antibiotic erythromycin (ERYr) and tetracycline (TETr) resistant enterococci, associated resistance genes and species distribution in a cow-calf production system. In 2017 and 2018, 32 Angus breed cow-calf pairs were randomly assigned to feed on tall fescue or wheat pasture in two independent field experiments. During the grazing experiments of 2-3 weeks, fecal samples were collected weekly and cultured to enumerate, isolate and identify ERYr, TETr, and generic enterococci, using media supplemented with erythromycin, tetracycline or non-supplemented media, respectively. The two main species frequently associated with human illnesses, Enterococcus faecium and E. faecalis, were widely distributed in the cow-calf groups. Generic and TETr- enterococci were prevalent (96-100% prevalence) and abundant (3.2-4.9 log10 CFU/g) in the cow-calf population; however, ERYr enterococci were enumerable by direct plating only from a single cow despite being detected in at least 40% of the fecal samples after enrichment, showing their low abundance. TET- and ERY-resistance were mainly conferred by tet(M) and erm(B), respectively. Wheat grazing reduced the concentration of TETr enterococci and modified enterococcal species and resistance gene distributions. Hence, it is necessary to further investigate wheat grazing in cow-calf production as a potential strategy to mitigate antimicrobial resistance.

Shifting the distribution: modulation of the lipid profile in Yarrowia lipolytica via iron content.

Microbial fermentation offers a sustainable source of fuels, commodity chemicals, and pharmaceuticals, yet strain performance is influenced greatly by the growth media selected. Specifically, trace metals (e.g., iron, copper, manganese, zinc, and others) are critical for proper growth and enzymatic function within microorganisms yet are non-standardized across media formulation. In this work, the effect of trace metal supplementation on the lipid production profile of Yarrowia lipolytica was explored using tube scale fermentation followed by biomass and lipid characterization. Addition of iron (II) to the chemically defined Yeast Synthetic Complete (YSC) medium increased final optical density nearly twofold and lipid production threefold, while addition of copper (II) had no impact. Additionally, dose-responsive changes in lipid distribution were observed, with the percent of oleic acid increasing and stearic acid decreasing as initial iron concentration increased. These changes were reversible with subsequent iron-selective chelation. Use of rich Yeast Peptone Dextrose (YPD) medium enabled further increases in the production of two specialty oleochemicals ultimately reaching 63 and 47% of the lipid pool as α-linolenic acid and cyclopropane fatty acid, respectively, compared to YSC medium. Selective removal of iron (II) natively present in YPD medium decreased this oleochemical production, ultimately aligning the lipid profile with that of non-supplemented YSC medium. These results provide further insight into the proposed mechanisms for iron regulation in yeasts especially as these productions strains contain a mutant allele of the iron regulator, mga2. The work presented here also suggests a non-genetic method for control of the lipid profile in Y. lipolytica for use in diverse applications. KEY POINTS: • Iron supplementation increases cell density and lipid titer in Yarrowia lipolytica. • Iron addition reversibly alters lipid portfolio increasing linolenic acid. • Removal of iron from YPD media provides a link to enhanced oleochemical production.

Transgenerational effects on development following microplastic exposure in Drosophila melanogaster.

BackgroundPlastic pollution affects all ecosystems, and detrimental effects to animals have been reported in a growing number of studies. However, there is a paucity of evidence for effects on terrestrial animals in comparison to those in the marine realm.MethodsWe used the fly Drosophila melanogaster to study the effects that exposure to plastics may have on life history traits and immune response. We reared flies in four conditions: In media containing 1% virgin polyethylene, with no chemical additives; in media supplemented with 1% or 4% polyvinyl chloride, known to have a high content of added chemicals; and control flies in non-supplemented media. Plastic particle size ranged from 23–500 µm. We studied fly survival to viral infection, the length of the larval and pupal stage, sex ratios, fertility and the size of the resultant adult flies. We then performed crossings of F1 flies in non-supplemented media and looked at the life history traits of the F2.ResultsFlies treated with plastics in the food media showed changes in fertility and sex ratio, but showed no differences in developmental times, adult size or the capacity to fight infections in comparison with controls. However, the offspring of treated flies reared in non-supplemented food had shorter life cycles, and those coming from both polyvinyl chloride treatments were smaller than those offspring of controls.

An image analysis method to quantify heterogeneous filamentous biomass based on pixel intensity values – Interrelation of macro- and micro-morphology in Actinomadura namibiensis

• An image analysis method for the heterogeneous cell morphology was developed. • Quantification of the network of dispersed mycelia. • Salt-enhanced cultivation affected pellet size and non-agglomerated hyphal network. • Hyphal network spacing was found to decrease during salt-enhanced cultivation. • A new correlation between macro- and micro-morphological parameters was found. The cell morphology of the filamentous actinobacterium Actinomadura namibiensis was evaluated by image analysis of microscopic pictures. A pixel intensity thresholding method was used to differentiate between pelleted bioagglomerates, the macro-morphology, and the characterization of individual hyphae, the micro-morphology. At both scales morphological differences between salt-enhanced cultivation (addition of 50 mM (NH 4 ) 2 SO 4 ), which increased the labyrinthopeptin A1 concentration from < 50–325 mg L −1 , and the non-supplemented standard medium were revealed. The major focus in this investigation was set on the analysis of the network of dispersed mycelia to provide complementary data to previously published pellet shape characteristics. Hyphal network spacing (HNS), a newly introduced shape descriptor to describe the spatial proximity of loosely entangled hyphae, was found to decrease to during salt-enhanced cultivation resulting in a 20 % lower HNS value compared to the unsupplemented control after 10 d of shaking flask cultivation. The macro-morphological pellet shape descriptors of circularity and aspect ratio correlated with individual micro-morphological parameters. The strongest correlation was found between pellet circularity and hyphal growth unit (HGU) (r = 0.73) and between pellet circularity and HNS (r = 0.82).

The enhanced biomass and lipid accumulation in algae with an integrated treatment strategy by waste molasses and Mg2+ addition

ABSTRACTThe effect of Mg2+ addition to waste molasses-based medium for algae growth on lipid accumulation and fatty acids profiles was studied. Further, the removal rate of residual metal ions in the culture medium under different concentrations of Mg2+ was also explored. When 800 μM Mg2+ was added to the medium, the peak values for biomass and lipid productivity were 1.81- and 2.28-times higher than the respective values for non-supplemented medium, exhibited a positive effect on biomass yield and lipid accumulation. The fatty acid profile of algae grown in the optimized medium revealed higher monounsaturated fatty acid levels than that grown in control medium. Furthermore, the biodiesel properties calculated by fatty acid profiles were also discussed. In addition, residual metal ions and nutrient (COD, TN, TP) in waste molasses were efficiency removed upon the addition of Mg2+. The results provide a strategy for utilizing magnesium ions as an additive to waste molasses-based medium for algae growth, producing profitable biodiesel and consuming industrial waste.

Comparison between curcumin and all-trans retinoic acid in the osteogenic differentiation of mouse bone marrow mesenchymal stem cells.

The use of bone marrow mesenchymal stem cells (BMSCs) has great potential in cell therapy, particularly in the orthopedic field. BMSCs represent a valuable renewable cell source that have been successfully utilized to treat damaged skeletal tissue and bone defects. BMSCs can be induced to differentiate into osteogenic lineages via the addition of inducers to the growth medium. The present study examined the effects of all-trans retinoic acid (ATRA) and curcumin on the osteogenic differentiation of mouse BMSCs. Morphological changes, the expression levels of the bone-associated gene markers bone morphogenetic protein 2, runt-related transcription factor and osterix during differentiation, an in vitro mineralization assay, and changes in osteocalcin expression revealed that curcumin supplementation promoted the osteogenic differentiation of BMSCs. By contrast, the application of ATRA increased osteogenic differentiation during the early stages, but during the later stages, it decreased the mineralization of differentiated cells. In addition, to the best of our knowledge, the present study is the first to examine the effect of curcumin on the osteogenic potency of mouse embryonic fibroblasts (MEFs) after reprogramming with human lim mineralization protein (hLMP-3), which is a positive osteogenic regulator. The results revealed that curcumin-supplemented culture medium increased hLMP-3 osteogenic potency compared with that of MEFs cultured in the non-supplemented medium. The present results demonstrate that enrichment of the osteogenic culture medium with curcumin, a natural osteogenic inducer, increased the osteogenic differentiation capacity of BMSCs as well as that of MEFs reprogrammed with hLMP-3.

Differential gene expression of a feed-spiked super-producing CHO cell line

Feed supplements are concentrated cell culture media that contain a variety of nutrients, which can be added during a bioprocess. During fed-batch cultivation, feed media are typically added to a growing cell culture to maximize cell and product concentrations. In this study, only a single shot of feed medium was added on day 0 to a basal cell culture medium and compared to non-supplemented basal medium (feed-spiked at day 0 versus control experiments) by cultivation of a recombinant mAb expressing CHO cell line in batch mode under controlled conditions in a bioreactor. Since the feed-spike at day 0 was based on existing medium components without introducing additional supplements, a desirable process with decreased complexity was generated. Unlike cells in basal medium, feed-spiked cultures reached almost 2× higher peak cell concentrations (10 × 106 c/mL vs. 18 × 106 c/mL) and 3× higher antibody concentrations (0.8 g/L vs. 2.4 g/L). Batch process time and the integral over the viable cell count were similar for both process types. Constantly high cell-specific production rates in feed-spiked cultures (70 pg/cell/day) compared to continuously declining rates in basal medium (from 70 to 10 pg/cell/day) were responsible for an overall 70% higher cell-specific production rate and the higher product concentrations. To associate gene expression patterns to different process proceedings, transcriptome analysis was performed using microarrays. Several transcripts that are involved with glutamine de novo synthesis and citric acid cycle were significantly upregulated on several days in feed-spiked cultures. The top identified gene ontology (GO) terms related well to cell cycle and primary metabolism, cellular division as well as nucleobase formation or regulation, which indicated a more active proliferative state for feed-spiked cultures. KEGG biochemical pathway analysis and Gene set enrichment analysis (GSEA) further confirmed these findings from a complementary perspective. Moreover, several interesting gene targets, which have not yet been associated with recombinant protein expression, were identified that related to a higher proliferative state, growth, protein synthesis, cell-size control, metabolism, cell survival as well as genes that are associated with the control of the mammalian target of rapamycin (mTOR) in feed-spiked cultures. Analysis of critical product quality attributes (i.e. glycosylation, charge variants and size distribution) showed that feed-spiking did not change antibody quality.

Embryotropic effects of vascular endothelial growth factor on porcine embryos produced by in vitro fertilization

Current research suggests that supplementing in vitro culture (IVC) media with vascular endothelial growth factor (VEGF) may have beneficial effects on the development of porcine embryos in vitro. However, the molecular signaling mechanisms underlying this effect are unclear. Therefore, we aimed to investigate the effects of VEGF on molecular signaling events during in vitro embryonic development of porcine embryos. Porcine oocytes matured in vitro were fertilized, and the resultant zygotes were cultured with 5 ng/mL of VEGF supplemented with or without fetal bovine serum from day 4 till day 7. Without VEGF and/or FBS served as the control group. Real-time quantitative PCR was used to detect expression patterns of apoptosis- and oxidative stress-related genes in day 7 blastocysts (BLs). Early-stage apoptosis was detected by annexin-V assays in day 2 and day 7 embryos. We found that the addition of VEGF throughout the culture period with or without FBS supplementation significantly improved embryo survival and development. Supplementation with VEGF in the IVC medium significantly increased early BL formation (p < 0.05), although addition of FBS on day 4 significantly increased hatched BL formation (p < 0.05) regardless of VEGF supplementation. However, supplementation of media with both VEGF and FBS increased the formation of expanded BLs synergistically. The average total cell numbers per BL were significantly (p < 0.05) higher in embryos supplemented with VEGF and FBS than in those supplemented with either VEGF or FBS alone. We also found that accumulation of reactive oxygen species in VEGF-treated embryos was significantly lower (p < 0.05) than that in untreated embryos. The mRNA levels of caspase-3 were significantly lower (p < 0.05), and those of Bcl-2 and Nrf-2 were significantly higher (p < 0.05) in embryos grown in VEGF-supplemented media than in embryos grown in non-supplemented media. Furthermore, on day 2, the numbers of viable embryos (44.06 ± 3.94%) and blastomeres (67.18 ± 3.60%) were significantly higher (p < 0.05), and the numbers of early apoptotic embryos (55.94 ± 3.94) and blastomeres (23.23 ± 4.22) were significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Furthermore, the numbers of early apoptotic cells in BLs on day 7 were also significantly lower (p < 0.05) in VEGF-treated BLs than in controls. Overall, our results indicate that supplementing IVC media with VEGF during in vitro culture of porcine embryos increases their developmental potential.

Impact of supplementation of semen extender with antioxidants on the quality of chilled or cryopreserved Arabian stallion spermatozoa

The aim of the present study was to evaluate the effects of supplementation of semen extender with various non-enzymatic antioxidants on the quality of cooled or cryopreserved Arabian stallion spermatozoa. Semen collected from four pure Arabian stallions was centrifuged at 600g for 15 min. Spermatozoa were then diluted in INRA-82 extender supplemented with bovine serum albumin (BSA; 0, 10, 15 and 20 mg/mL) or trehalose (0, 75, 100 and 150 mM) or zinc sulphate (0, 100, 150 and 200 μM). The diluted semen was then either cooled at 5 °C or cryopreserved in 0.5–ml plastic straws. After cooling or thawing, sperm motility, viability, sperm abnormalities, viability index, and plasma membrane integrity were evaluated. The results showed that supplementation of semen extender with 150 mM trehalose or with 200 μM zinc sulphate significantly (P < 0.05) improved motility, viability, sperm membrane integrity and acrosome status in Arabian stallion spermatozoa after cooling or after freezing and thawing compared with controls (non-supplemented media) or with those supplemented with other concentrations of trehalose or zinc sulphate. Supplementation of semen extender with BSA did not improve sperm motility or cryosurvival of Arabian stallion spermatozoa after cooling or after freezing and thawing. In conclusion, supplementation of semen extender with non-enzymatic antioxidants (trehalose or zinc sulphate) improved the quality of chilled and frozen/thawed Arabian stallion spermatozoa. The most beneficial effects occur when semen diluent was supplemented with 150 mM trehalose or 200 μM zinc sulphate.

Antimicrobial-Resistant Escherichia coli Survived in Dust Samples for More than 20 Years.

In a retrospective study, 119 sedimentation dust samples stored between five and 35 years from various barns of intensive livestock farming were evaluated for the occurrence of cultivatable Escherichia coli. Growth of E. coli occurred in 54 samples. Successful cultivation was achieved in samples from as early as 1994. The frequency of detection increased from earlier to later time periods, but the concentrations, which ranged between 3.4 × 102 and 1.1 × 105 colony-forming units per gram, did not correlate with sample age (Spearman rank correlation; p > 0.05). We hypothesize that E. coli cells survived in dust samples without cell division because of the storage conditions. Dry material (dust) with low water activities (arithmetic mean < 0.6) and storage at 4°C in the dark likely facilitated long-term survival. E. coli were isolated on MacConkey agar with and without ciprofloxacin supplementation. For 110 isolates (79 from non-supplemented media and 31 from supplemented media), we determined the E. coli phylotype and antimicrobial resistance. Six phylogenetic groups were identified. Phylogroups A and B1 predominated. Compared to group A, phylogroup B1 was significantly associated with growth on ciprofloxacin-supplemented media (chi-square test, p = 0.003). Furthermore, the antibiotic resistance profiles determined by a microdilution method revealed that isolates were phenotypically resistant to at least one antimicrobial substance and that more than 50% were resistant to a minimum of five out of 10 antibiotics tested. A linear mixed model was used to identify factors associated with the number of phenotypic resistances of individual isolates. Younger isolates and isolates from fattening poultry barns tended to be resistant to significantly more antibiotics than older isolates and those from laying-hen houses (p = 0.01 and p = 0.02, respectively). Sample origin and storage conditions may have influenced the number of antimicrobial resistances. Overall, we found that under particular conditions, dust from farm animal houses can be reservoirs for antimicrobial-resistant E. coli for at least 20 years. The survival strategies that allow E. coli to survive such long periods in environmental samples are not fully understood and could be an interesting research topic for future studies.

Supplemented base medium containing Amburana cearensis associated with FSH improves in vitro development of isolated goat preantral follicles

The effects of Amburana cearensis ethanolic extract, with or without addition of a mix of supplements associated or not with FSH, on in vitro morphology and development of caprine secondary follicles were evaluated. In experiment 1, isolated follicles (250 μm in diameter) were cultured for 12 days in alpha-modified minimal essential medium (α-MEM) alone (control) or in medium composed of different concentrations of A. cearensis extract (Amb 0.1; 0.2, or 0.4 mg/mL). In experiment 2, culture media were α-MEM or Amb 0.2 mg/mL (both without supplements), or these same media supplemented with BSA, insulin, transferrin, selenium, glutamine, hypoxanthine, and ascorbic acid (referred as α-MEM+ and Amb 0.2+, respectively), or these last groups also supplemented with sequential FSH (100 ng/mL from Day 0 to Day 6; 500 ng/mL from Day 6 to Day 12), constituting groups α-MEM+ + FSH and Amb 0.2+ + FSH. At the end of culture in experiment 1, control medium (α-MEM) and Amb 0.2 mg/mL had higher percentages (P < 0.05) of morphologically normal follicles and percentage of fully grown oocytes, i.e., oocyte greater than 110 μm, compared to the other A. cearensis extract concentrations. In experiment 2, all supplemented media had higher percentages (P < 0.05) of normal follicles and antrum formation than nonsupplemented media. In addition, follicles cultured in Amb 0.2+ + FSH showed an average increase in diameter higher (P < 0.05) than the other treatments. Oocytes cultured in both treatments supplemented with FSH showed greater glutathione and active mitochondria levels than nonsupplemented media but similar to the other treatments. In conclusion, A. cearensis extract (0.2 mg/mL) added by supplements and FSH improves follicular growth. Therefore, it can be an alternative culture medium for goat preantral follicle development.

Calcium transcriptionally regulates the biofilm machinery of Xylella fastidiosa to promote continued biofilm development in batch cultures.

The functions of calcium (Ca) in bacteria are less characterized than in eukaryotes, where its role has been studied extensively. The plant-pathogenic bacterium Xylella fastidiosa has several virulence features that are enhanced by increased Ca concentrations, including biofilm formation. However, the specific mechanisms driving modulation of this feature are unclear. Characterization of biofilm formation over time showed that 4 mM Ca supplementation produced denser biofilms that were still developing at 96 h, while biofilm in non-supplemented media had reached the dispersal stage by 72 h. To identify changes in global gene expression in X. fastidiosa grown in supplemental Ca, RNA-Seq of batch culture biofilm cells was conducted at three 24-h time intervals. Results indicate that a variety of genes are differentially expressed in response to Ca, including genes related to attachment, motility, exopolysaccharide synthesis, biofilm formation, peptidoglycan synthesis, regulatory functions, iron homeostasis, and phages. Collectively, results demonstrate that Ca supplementation induces a transcriptional response that promotes continued biofilm development, while biofilm cells in nonsupplemented media are driven towards dispersion of cells from the biofilm structure. These results have important implications for disease progression in planta, where xylem sap is the source of Ca and other nutrients for X. fastidiosa.

Impact of the administration of a third-generation cephalosporin (3GC) to one-day-old chicks on the persistence of 3GC-resistant Escherichia coli in intestinal flora: An in vivo experiment

The aim of the experiment was to evaluate under controlled conditions the impact on the excretion of 3GC-resistant Escherichia coli of the injection of one-day-old chicks with ceftiofur, a third-generation cephalosporin (3GC). Three isolators containing specific-pathogen-free chicks were used. In the first one, 20 birds were injected with ceftiofur then ten of them were orally inoculated with a weak inoculum of a 3GC-resistant E. coli field isolate containing an IncI1/ST3 plasmid encoding a blaCTX-M-1 beta-lactamase. The other chicks were kept as contact birds. None of the 20 birds in the second isolator were injected with ceftiofur, but ten of them were similarly inoculated with the 3GC-resistant strain and the others kept as contact birds. A third isolator contained ten non-injected, non-inoculated chicks. Fecal samples were collected regularly over one month and the E. coli isolated on non-supplemented media were characterized by antimicrobial agar dilution, detection of selected resistance genes and determination of phylogenetic group by PCR. The titers of 3GC-resistant E. coli in individual fecal samples were evaluated by culturing on 3GC-supplemented media. Results showed that the inoculated strain rapidly and abundantly colonized the inoculated and contact birds. The ceftiofur injection resulted in significantly higher percentages of 3GC-resistant E. coli isolates among the analyzed E. coli. No transfer of the 3GC-encoding plasmid to other isolates could be evidenced. In conclusion, these results highlight the dramatic capacity of 3GC-resistant E. coli to colonize and persist in chicks, and the selecting pressure imposed by the off-label use of ceftiofur.

Rooting response of Prunus domestica L. microshoots in the presence of phytoactive medium supplements

The study aimed at evaluating the response of common plum (Prunus domestica L.) microshoots during in vitro rooting in the presence of two phytoactive medium supplements, i.e. a dialyzate of pineapple pulp and a conditioned medium containing green algae Desmodesmus subspicatus exudates. Rooting efficiency was evaluated after 4 weeks of culture. During the root induction phase the content of phenolic compounds in shoot bases was determined and anatomical studies were conducted. Medium supplements were analyzed for the content of carbohydrates and phenolic acids. Both supplements were efficient in rooting induction of shoots of a difficult-to-root cultivar ‘Wegierka Zwykla’. Medium supplementation allowed a significant reduction in the exogenous auxin content required for rooting. The highest rooting efficiencies on supplemented media were 28.9 and 27.8 %, in comparison with 33.3 % obtained in the control medium with doubled concentration of exogenous auxins. In the easy-to-root plum cultivar ‘Wegierka Dąbrowicka’ the rooting rate was slightly reduced in the presence of pineapple dialyzate, while in the presence of algal conditioned medium the rooting rate decreased substantially compared with the non-supplemented medium. Approximately 30 % of ‘Wegierka Dąbrowicka’ shoots rooted on supplemented auxin-free media. The content of phenolic compounds accumulated in shoot bases during the root induction stage reflected the differences in rooting ability between both plum cultivars, indicating potential stressful conditions of the culture generated by the presence of phytoactive natural supplements. Anatomical study allowed to recognize the mode of dedifferentiation leading to adventitious rhizogenesis in the common plum. The results are discussed in relation to the composition of medium supplements and their potential root-promoting activity.

Evaluation of the Antithrombin Potential in a Thrombin Generation Assay Performed at the Surface of Endothelial Cells

Thrombin generation assay (TGA) was recently evaluated on a living endothelial-derived cell line (Coll et al. J. Thromb. Haemost. 2013; 11, 1916). This innovative assay brought into an hemostasis assay the cellular components of the anticoagulation pathway (APC and TFPI pathways) as well as a activated cell surface. It might help elucidate the relationship between hemostasis and inflammation in a more complex system.In the aim of evaluating the potential of antithrombin (AT) connecting both processes we set-up a similar assay on human vein endothelial cells (HUVEC). We first demonstrated that thrombin generation can be measured in flat-bottom 96-wells in factor IX-or factor VIII-deficient plasma substituted by either 0.1 or 1 U/ml of FIX or FVIII, respectively. Next, HUVEC were grown and expanded in a complete commercial medium (EndoGRO-LS, Millipore) for no more than 6 passages. Wells were then coated with gelatin 1% and cells seeded at 10,000 cells/well. The binding of plasma-derived AT (Aclotine ®, LFB; France dialyzed in cell culture medium) to HUVEC was demonstrated as being dose- (0.5; 1; 2.5 and 5 U/ml) and time- (0-6 hours) dependent. Saturating conditions were found using 2.5 U/ml AT for a 2h incubation. We also showed that the binding was moderately affected in the presence of heparin at concentrations up to 50 U/ml (loss of 19% of the signal) and not at all following an heparanase I+II+III treatment suggesting that another receptor(s) than cellular heparan sulfates being responsible for this interaction.The effect of AT on coagulation was then compared in the presence of cells or not. To do this cells were grown to confluence, washed with non-supplemented medium and incubated in the presence of the TGA mix (plasma containing AT or not, 0.5 pM Tissue Factor, 4 µM Phospholipids). The reaction was initiated by injection of the FluCa kit thrombin substrate (Stago). In the presence of HUVEC, the efficiency of thrombin generation from a control plasma (Unicalibrator, Stago) was decreased with a lag time increased (from 5.67 min to 6.83 min), the peak height diminished from 204.4 nM thrombin to 150.4 nM and the velocity from 55.8 nM/min to 33.4 nM/min. However, the overall amount of thrombin generated was less affected, diminishing from 1515.5 nM to 1482 nM. These data confirms that the presence of the HUVEC anticoagulants pathways can effectively diminish the thrombin generation.Without cells, the presence of 0.5, 1 or 2 U/ml AT dose-dependently decreased the generation of thrombin from the control plasma. The velocity was decreased by 23.2%, 57.6% and 75.5% and the peak height by 33.5%, 61.5% and 78.8%, respectively. When the same experiment was performed in the presence of HUVEC cells, the concentrations of AT similarly decreased the velocity by 34.2%, 54% and 70 % and the peak height by 39%, 59.1% 74.3%, respectively. There was no difference in the TGA parameters if AT was pre-incubated at the surface of the cells for up to 2h prior the TGA or if it was added extemporaneously. These results indicate that the presence of HUVEC did not modulate the in vitro effect of AT during coagulation. The effect of AT on the cell response during this process are in the process of being investigated with a particular focus on the anti-inflammatory properties of AT. DisclosuresCatieau:LFB Biotechnologies: Employment. Chtourou:LFB Biotechnologies: Employment. Plantier:LFB Biotechnologies: Employment.