Listeria monocytogenes sensitivity to antimicrobial treatments depends on cell origin

In this study we investigated how cell origin could affect the efficacy of an antimicrobial treatment (mild heating combined with terpenoids) in Listeria monocytogenes Scott A, considering cells from: 1. single colony, 2. glycerol stock, 3. cold adapted culture, and 4. fresh culture in stationary phase. After treatment, culturability on BHI medium and viability assessed by flow cytometry were evaluated. Our results showed that the cell origin significantly impacted viability and culturability of L. monocytogenes towards antimicrobial treatment. The mild heat treatment combined or not with terpenoids mainly affected culturability rather than viability, although the culturability of cells from single colony was less impacted. Therefore, to mimic the worst scenario, these latter were selected to contaminate Gorgonzola rind and roast beef slices and we evaluated the ability of L. monocytogenes cells to recover their culturability (on ALOA agar medium) and to growth on the food matrix stored at 4 °C for 7 days. Our results suggest that only Gorgonzola rind allowed a partial recovery of the culturability of cells previously heated in presence or not of terpens. In conclusion, we found a connection between the cell history and sensitivity toward an antimicrobial treatment, underlying the importance to standardize the experimental procedures (starting from the cells to be used in the assay) in the assessment of cell sensitivity to a specific treatment. Finally, our study clearly indicated that VBNC cells can resuscitate under favorable conditions on a food matrix, becoming a threat for consumer’s health.

Temporal Evolution of the Gravitaxis of Euglena gracilis from a Single Cell

Gravitaxis is one of the most important issues in the growth of microalgae in the water column; it determines how easily cells receive sunlight with a comfortable intensity that is below the damaging threshold. We quantitatively investigated and analyzed the gravitaxis and cell multiplication of Euglena gracilis using vertically placed microchambers containing a single cell. A temporal change in gravitaxis and cell multiplication was observed after transferring the cells to fresh culture medium for 9 days. We performed 29 individual experiments with 2.5 × 2.5 × 0.1 mm square microchambers and found that the cells showed positive, negative, and moderate gravitaxis in 8, 7, and 14 cases, respectively, after transferring to fresh culture medium. A common trend was observed for the temporal change in gravitaxis for the eight initially positive gravitaxis cases. The cells with initially positive gravitaxis showed a higher rate of cell multiplication than those with initially negative gravitaxis. We also discussed the gravitaxis mechanism of E. gracilis from the observed trend of gravitaxis change and swimming traces. In addition, bioconvection in a larger and thicker chamber was investigated at a millimeter scale and visualized.

Induction of Hairy Roots on Somatic Embryos of Rhizoclones from Typha domingensis Seedlings

A protocol for the induction of hairy roots on somatic embryos of rhizoclones from Typha domingensis seedlings grown in hydroponic rhizotron systems was established for the first time. Rhizogenesis was induced through the agrotransformation of somatic embryos in oblong and scutellar states of development using the K599, LBA9402, and A4 strains of Agrobacterium rhizogenes. The transfection to the embryos was performed by cocultivation of rhizoclones on a Murashige and Skoog mineral medium at 50% strength (MS0.5), in the dark, at 28 ± 2 °C for 72 h. In contrast to nontransformed embryos that did not exhibit any root tissue, transformed embryos presented hairy roots that varied in number, length, and density depending on the bacterial strain, and K599 was the most effective strain. After analysis via optical microscopy, the transformed embryos were collected and transferred to fresh culture media supplemented with 400 mg mL−1 cefotaxime and 10 mg L−1 ascorbic acid. The efficiency of transformation and survival of the oblong and scutellar embryos were similar among the three bacterial strains. The results show that agrotransformation of somatic embryos of rhizoclones from T. domingensis is a novel and viable strategy for the generation of genetic transformants of Typha that have potential applications in bioremediation technologies.

Complete Genome Sequence of Paenibacillus sp. Strain E222, a Bacterial Symbiont of an Epichloë Fungal Endophyte of Ryegrass

ABSTRACT We report on the whole-genome sequence of Paenibacillus sp. strain E222, a bacterium isolated from a fresh culture of Epichloë festucae var. lolii, a mutualistic fungal endophyte of perennial ryegrass. The genome has a size of 7.8 Mb and a G+C content of 46% and encodes 6,796 putative protein-coding genes.

Autochtonous Strain Enterococcus faecium EF2019(CCM7420), Its Bacteriocin and Their Beneficial Effects in Broiler Rabbits—A Review

The present review evaluates and compares the effects achieved after application of rabbit-derived bacteriocin-producing strain Enterococcus faecium CCM7420 with probiotic properties and its bacteriocin Ent7420. The experiments included varying duration of application (14 and 21 days), form of application (fresh culture and lyophilized form), combination with herbal extract and application of the partially purified enterocin—Ent7420, produced by this strain. Results from these studies showed that E. faecium CCM7420 strain was able to colonize the gastrointestinal tract (caecum) of rabbits (in the range < 1.0–6.7 log cycle, respectively 3.66 log cycle on average), to change the composition of intestinal microbiota (increased lactic acid bacteria, reduced counts of coliforms, clostridia and staphylococci), to modulate the immunity (significant increase of phagocytic activity), morphometry (enlargement absorption surface in jejunum, higher villi height:crypt depth (VH:CD) ratio), physiological (serum biochemistry; altered total proteins, glucose and triglycerides levels) and parasitological (Eimeria sp. oocysts) parameters and to improve weight gains (in the range 4.8–22.0%, respectively 11.2% on average), feed conversion ratio and meat quality (physicochemical traits and mineral content).

BACTERIAL AND FUNGAL CONTAMINANTS OF TISSUE-CULTURED 'LAKATAN' BANANA

This study aimed to characterize the bacterial and fungal contaminants of tissue-cultured 'Lakatan' banana (Musa acuminata) during initiation stage. This was conducted at the University of Southeastern Philippines, Tagum-Mabini Campus from October 2015 to February 2016. The sterilized banana explant was placed in a bottle of solidified nutrient agar medium. Detectable bacterial and fungal contaminants were isolated and sub-cultured into the fresh medium two to four days after incubation. The bacterial isolates were cultivated by streaking into fresh culture medium and incubated at 0 0 30-32 C for three days and fungi culture disk was transferred to fresh culture medium and incubated at 30-32 C for three to five days. Both bacterial and fungal contaminants were identified and characterized and assessed for extent of contamination. Results showed that the different contaminants occurred during the initiation stage of tissue-cultured 'Lakatan' banana meriplants were composed of Rhizopus sp., unidentified fungus and Gram-negative bacterium. Generally, 35% contamination was observed on this stage.

DISKURSUS THE MALAYS DAN MALAYNESS : PERSPEKTIF HERMENEUTIKA FILOSOFIS GADAMER

This paper aims to discuss informatively and critically-philosophically the two main discourses, namely The Malays and Malayness, which in the context of history and the dynamics of Malay culture are suggested to overlap, so that it is often misunderstood. The use of Gadamer philosophical hermeneutics as a media in analyzing data in this study is expected to be able to find new understanding of the two themes above, especially in the context of strengthening values and forming culture-based characters. The scrutiny and review of the treasures of Malay culture found that the Malay culture which stretched in a span of three periods, the past-present-future found a starting point as a place for cultural assessment, especially after the entry and development of Islam in the Malay-Indonesian Archipelago. The intended point is the Malay Culture "which is called the fresh culture. "Culture Appearance" takes place continuously and tracks in almost every practice of life without negating the old belief patterns (customs and traditions) that first enter and come into contact with the dynamics of people's lives.

In vivo and in vitro strategies to support caprine preantral follicle development after ovarian tissue vitrification

The aim of the present study was to compare fresh and vitrified goat ovarian tissue after autotransplantation and in vitro culture. Adult goats were completely ovariectomised and each ovarian pair was sliced and distributed among six different treatment groups: fresh control, fresh transplant, fresh culture, vitrified control, vitrified transplant and vitrified culture. Follicular morphology, development, growth, density, revascularisation and hormone production were evaluated in all groups. Three antral follicles (two in the fresh transplant and one in the vitrified transplant groups) were observed on the surface of the graft 90 days after transplantation. The percentage of morphologically normal follicles was similar in the fresh control, fresh transplant and vitrified transplant groups. The percentage of developing (transition, primary and secondary) follicles was higher after in vitro culture of fresh or vitrified tissue. Transplantation resulted in a lower follicle density. Serum oestradiol concentrations remained constant during the entire transplantation period. In contrast, progesterone production decreased significantly. Expression of CD31 mRNA was lower in fresh culture. In conclusion, restoration of goat ovarian function can be successfully achieved following transplantation of both fresh and vitrified goat ovarian tissue. However, transplantation induced higher follicle loss than in vitro culture.

Adhesion of Sickle RBCs to Heme-Activated Endothelial Cells Correlates with Patient Clinical Phenotypes

Severe hemolysis and associated high levels of hemolytic biomarkers, including LDH and heme, are among major constituents of the pathophysiology of sickle cell disease (SCD). Elevated extracellular heme due to hemolysis overwhelms endogenous detoxification mechanisms and leads to oxidative stress, triggering systemic endothelium activation, vascular dysfunction, and end organ damage. To understand the role of red blood cells (RBC) in this process, we assessed sickle RBC adhesion to heme-activated endothelial cells utilizing an endothelialized microfluidic platform in a clinically diverse patient population. Human umbilical vein endothelial cells (HUVECs) were seeded into fibronectin (FN) functionalized microfluidic channels and incubated for 4 hours in a 37°C and 5% CO2 environment. Next, the confluent monolayers were loaded and incubated for 45 minutes with fresh culture medium or at one of two concentrations of heme solutions: (1) 20 μM that corresponds to physiological heme levels in SCD patients, and (2) 40 μM. SCD blood samples, collected from 8 patients (7 HbSS and 1 HbS/β0 thal; 3 males and 5 females), were centrifuged to remove the plasma and washed with PBS thrice prior to flow experiments. RBCs were re-suspended in culture medium at a hematocrit of 25%. A total volume of 15 µl RBC suspension was perfused into the microchannels followed by rinsing with fresh culture medium at 1 dyne/cm2, which corresponds to the typical shear stress value observed in post-capillary venules. The fully closed and hermetically sealed microfluidic system design ensured the stability of gas composition in the culture medium during the experiments. Endothelialized microchannels supported sickle RBC adhesion to non-treated, 20 µM heme-activated, and 40 µM heme-activated HUVECs (Fig. 1A, B, C). Adhesion results suggested that activation of HUVECs by heme mediated RBC adhesion in a concentration-dependent manner, with a significant difference observed at 40 µM (Fig. 1D, paired t-test, p&lt;0.05). Notably, a heterogeneous heme-mediated adhesion profile was seen among patients. Sickle RBC adhesion to 20 μM heme-activated HUVECs was significantly increased in patients with higher LDH levels (Fig. 1E, p=0.024, ANOVA), higher absolute reticulocyte counts (Fig. 1F, p=0.002, ANOVA), and lower total hemoglobin (Fig. 1G, p=0.016, ANOVA), which are indicative of elevated hemolysis. All patients in the high adhesion group (&gt;200 adherent RBCs) had elevations in serum LDH levels and in absolute reticulocyte counts. Moreover, patients with a recent transfusion had higher RBC adhesion to 40 µM heme-activated HUVECs compared to patients with no transfusion in the last 3 months (Fig. 1H, p&lt;0.05, ANOVA). Here, we report a direct link between heme-driven endothelial activation and RBC adhesion in a patient-specific manner. In patients with a more severe clinical phenotype, as reflected in LDH, total hemoglobin, and absolute reticulocyte or recent blood transfusions, we found greater RBC adhesion to heme-activated HUVECs. These findings suggest that RBCs from those patients most likely to experience hemolysis in vivo may also be those RBCs most likely to adhere to heme-activated endothelium. Acknowledgments: This work was supported by grants #2013126 and #2015191 from the Doris Duke Charitable Foundation, National Heart Lung and Blood Institute R01HL133574, and National Science Foundation CAREER Award 1552782. Figure 1: Sickle RBC adhesion to heme-activated HUVECs and clinical associations. Sickle RBCs adherent to immobilized HUVECs in (A) non-activated, (B) 20 µM heme-activated, and (C) 40 µM heme-activated microchannels. The microscope images illustrate a small portion of the full microchannel surface. (D) RBC adhesion to HUVECs increased depending on the heme concentration, with a significant difference at the 40 µM level (paired t test, p&lt;0.05). Patients with higher LDH (E) as well as absolute reticulocyte counts (F), and lower total hemoglobin (G) showed significantly greater RBC adhesion to 20 µM heme-activated HUVECs (ANOVA). (H) Furthermore, patients with a recent transfusion history displayed elevated RBC adhesion to 40 µM heme-activated HUVECs compared to non-transfused patients (ANOVA). The dashed rectangles indicate the normal clinical values for healthy individuals, while all patients had total hemoglobin levels lower than normal. Scale bars represent 30 µm. Figure 1 Figure 1. Disclosures Little: Hemex Health: Equity Ownership. Gurkan: Hemex Health: Employment, Equity Ownership.