Immune response and protection efficacy of formalin-killed vaccines against Streptococcus iniae in four-finger threadfin Eleutheronema tetradactylum.

Four-finger threadfin, Eleutheronema tetradactylum farming in southern Taiwan has been facing disease problems caused by Streptococcus iniae since 2018. The development of a vaccine against infectious S. iniae in the cultured threadfin industry is necessary. Thus, this study aimed to examine the efficacy of threadfin immunized formalin-killed cells (FKC) from S. iniae GSI-111 for 42 days post-vaccination (dpv) using two doses of FKC alone (a booster at 14 dpv) as group A, and FKC mixed with ISA763A adjuvant using a single dose as group B or double doses as group C. Immunoglobulin (Ig)-M was purified from threadfin, and rabbit anti-threadfin IgM polyclonal antibodies were used to detect antibody level in immunized fish; the vaccinated group A displayed higher levels at 3 dpv and all vaccinated treatments demonstrated high antibody levels between 14 and 42 dpv. All vaccine groups showed significantly higher values of lysozyme activity at 42 dpv compared with the control group; the vaccinated A group peaked at 14 dpv. The expression profiles of pro-inflammatory and immune-related genes, TNF-α, IL-12A, and C2 were upregulated at 3 dpv, while CD8A and chemokine receptor CXCR4 were upregulated at 42 dpv. Finally, the threadfins were challenged with S. iniae at 42 dpv. The average relative percent survival was 96% for vaccination A and B treatments, and 100% for vaccination C treatment. In summary, this study demonstrated that FKC vaccines whether formulated with an adjuvant could stimulate immune response and effective protect threadfins against S. iniae infection.

Influence of Dead Cells Killed by Industrial Biocides (BAC and DBNPA) on Biofilm Formation.

Industrial biocides aim to keep water systems microbiologically controlled and to minimize biofouling. However, the resulting dead cells are usually not removed from the water streams and can influence the growth of the remaining live cells in planktonic and sessile states. This study aims to understand the effect of dead Pseudomonas fluorescens cells killed by industrial biocides-benzalkonium chloride (BAC) and 2,2-dibromo-3-nitrilopropionamide (DBNPA)-on biofilm formation. Additionally, the effect of different dead/live cell ratios (50.00% and 99.99%) was studied. The inoculum was recirculated in a Parallel Plate Flow Cell (PPFC). The overall results indicate that dead cells greatly affect biofilm properties. Inoculum with DBNPA-dead cells led to more active (higher ATP content and metabolic activity) and thicker biofilm layers in comparison to BAC-dead cells, which seems to be linked to the mechanism of action by which the cells were killed. Furthermore, higher dead cell ratios (99.99%) in the inoculum led to more active (higher culturability, metabolic activity and ATP content) and cohesive/compact and uniformly distributed biofilms in comparison with the 50.00% dead cell ratio. The design of future disinfection strategies must consider the contribution of dead cells to the biofilm build-up, as they might negatively affect water system operations.

Mode of killing determines the necrotrophic response of oral bacteria.

Background: Bacteria respond to changes in their environment, such as nutrient depletion and antimicrobials exposure. Antimicrobials result not only in bacterial death, but also have a hand in determining species abundances and ecology of the oral biofilms. Proximity of dead bacterial cells to living ones is an important environmental change or stress factor. Dead bacteria represent high concentrations of nutrients, such as proteins, lipids, sugars, and nucleic acids. Living bacteria can use these biomasses as a nutrients source, which is termed necrotrophy. Aim: This study investigates the effect of exposing living oral bacteria (planktonic and biofilms) to their dead siblings after being killed by heat or hydrogen peroxide. Results: Tested bacterial species showed different responses towards the dead cells, depending on the mode of killing, the nutritional value of the culture media, and the the dead cells density. The multispecies oral biofilms showed different responses towards the supplementation of dead cells during biofilm development, while matured biofilms were more resilient. Conclusion: This study indicates that dead bacteria resulting from antiseptics use may imbalance the nutrient availability in the oral cavity, resulting in overgrowth of opportunistic species, and hence ecological changes in oral communities, or introducing new bacterial phenotypes.

Immune-stimulating Effect of Lactobacillus plantarum Ln1 Isolated from the Traditional Korean Fermented Food, Kimchi.

This study aimed to determine the immune-stimulating effects of heat-killed Lactobacillus plantarum Ln1 (HK-Ln1) through the production of nitric oxide (NO) and pro-inflammatory cytokine achieved by inducing NF-κB and mitogen-activated protein kinase (MAPK)-signaling pathways in macrophages. HK-Ln1 showed higher NO and cytokine production compared t°Control (nonstimulated lipopolysaccharide); in addition, the expression of inducible nitric oxide synthase (iNOS) was induced through HK-Ln1treatment. The phosphorylation of IκB-α and p65 increased following treatment by HK-Ln1, which implicates IκB-α degradation and the translocation of p65 to nucleus. In addition, the phosphorylation of MAPKs, ERK 1/2, JNK, and p38 was induced following HK-Ln1 treatment.

Image-based spatio-temporal model of drug delivery in a heterogeneous vasculature of a solid tumor - Computational approach.

The solute transport distribution in a tumor is an important criterion in the evaluation of the cancer treatment efficacy. The fraction of killed cells after each treatment can quantify the therapeutic effect and plays as a helpful tool to evaluate the chemotherapy treatment schedules. In the present study, an image-based spatio-temporal computational model of a solid tumor is provided for calculation of interstitial fluid flow and solute transport. Current model incorporates heterogeneous microvasculature for angiogenesis instead of synthetic mathematical modeling. In this modeling process, a comprehensive model according to Convection-Diffusion-Reaction (CDR) equations is employed due to its high accuracy for simulating the binding and the uptake of the drug by tumor cells. Based on the velocity and the pressure distribution, transient distribution of the different drug concentrations (free, bound, and internalized) is calculated. Then, the fraction of killed cells is obtained according to the internalized concentration. Results indicate the dependence of the drug distribution on both time and space, as well as the microvasculature density. Free and bound drug concentration have the same trend over time, whereas, internalized and total drug concentration increases over time and reaches a constant value. The highest amount of concentration occurred in the tumor region due to the higher permeability of the blood vessels. Moreover, the fraction of killed cells is approximately 78.87% and 24.94% after treatment with doxorubicin for cancerous and normal tissues, respectively. In general, the presented methodology may be applied in the field of personalized medicine to optimize patient-specific treatments. Also, such image-based modeling of solid tumors can be used in laboratories that working on drug delivery and evaluating new drugs before using them for any in vivo or clinical studies.

Designing primers and evaluation of the efficiency of propidium monoazide - Quantitative polymerase chain reaction for counting the viable cells of Lactobacillus gasseri and Lactobacillus salivarius.

The purpose of this study is to evaluate the efficiency of using propidium monoazide (PMA) real-time quantitative polymerase chain reaction (qPCR) to count the viable cells of Lactobacillus gasseri and Lactobacillus salivarius in probiotic products. Based on the internal transcription spacer and 23S rRNA genes, two primer sets specific for these two Lactobacillus species were designed. For a probiotic product, the total deMan Rogosa Sharpe plate count was 8.65±0.69 log CFU/g, while for qPCR, the cell counts of L. gasseri and L. salivarius were 8.39±0.14 log CFU/g and 8.57±0.24 log CFU/g, respectively. Under the same conditions, for its heat-killed product, qPCR counts for L. gasseri and L. salivarius were 6.70±0.16 log cells/g and 7.67±0.20 log cells/g, while PMA-qPCR counts were 5.33±0.18 log cells/g and 5.05±0.23 log cells/g, respectively. For cell dilutions with a viable cell count of 8.5 log CFU/mL for L. gasseri and L. salivarius, after heat killing, the PMA-qPCR count for both Lactobacillus species was near 5.5 log cells/mL. When the PMA-qPCR counts of these cell dilutions were compared before and after heat killing, although some DNA might be lost during the heat killing, significant qPCR signals from dead cells, i.e., about 4-5 log cells/mL, could not be reduced by PMA treatment. Increasing PMA concentrations from 100 µM to 200 µM or light exposure time from 5 minutes to 15 minutes had no or, if any, only minor effect on the reduction of qPCR signals from their dead cells. Thus, to differentiate viable lactic acid bacterial cells from dead cells using the PMA-qPCR method, the efficiency of PMA to reduce the qPCR signals from dead cells should be notable.

Heat-killed Lactobacillus spp. cells enhance survivals of Caenorhabditis elegans against Salmonella and Yersinia infections.

UNLABELLED: This study examined the effect of feeding heat-killed Lactobacillus cells on the survival of Caenorhabditis elegans nematodes after Salmonella Typhimurium and Yersinia enterocolitica infection. The feeding of heat-killed Lactobacillus plantarum 133 (LP133) and Lactobacillus fermentum 21 (LP21) cells to nematodes was shown to significantly increase the survival rate as well as stimulate the expression of pmk-1 gene that key factor for C. elegans immunity upon infection compared with control nematodes that were only fed Escherichia coli OP50 (OP50) cells. These results suggest that heat-killed LP133 and LF21 cells exert preventive or protective effects against the Gram-negative bacteria Salm. Typhimurium and Y. enterocolitica. To better understand the mechanisms underlying the LF21-mediated and LP133-mediated protection against bacterial infection in nematodes, transcriptional profiling was performed for each experimental group. These experiments showed that genes related to energy generation and ageing, regulators of insulin/IGF-1-like signalling, DAF genes, oxidation and reduction processes, the defence response and/or the innate immune response, and neurological processes were upregulated in nematodes that had been fed heat-killed Lactobacillus cells compared with nematodes that had been fed E. coli cells. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the feeding of heat-killed Lactobacillus bacteria to Caenorhabditis elegans nematodes was shown to decrease infection by Gram-negative bacteria and increase the host lifespan. C. elegans has a small, well-organized genome and is an excellent in vivo model organism; thus, these results will potentially shed light on important Lactobacillus-host interactions.

Application of ethidium bromide monoazide for quantification of viable and dead cells of Salmonella enterica by real-time loop-mediated isothermal amplification.

A rapid and efficient method for quantification and discrimination of Salmonella enterica ser. Enteritidis between viable and dead cells killed by heat was developed using ethidium bromide monoazide (EMA) in combination with a real-time loop amplified (Rti-LAMP) DNA assay. The use of 8.0 µg/ml or less of EMA did not inhibit DNA amplification in Rti-LAMP assays derived from viable cells. However, 8.0 µg/ml of EMA notably inhibited DNA amplification and significantly increased the Tp values with dead cells. When the DNA from 2000 viable CFU was subjected to EMA-Rti-LAMP the resulting Tp value was 13 min. In contrast, the DNA from 2000 CFU completely heat destroyed CFU still yielded a Tp value, which was greatly increased to 33.1 min. When the DNA from viable plus heat killed CFU at a ratio of 7:1993 was subjected to EMA-Rti-LAMP, the resulting Tp value was 19.3 min, which was statistically identical (P<0.05) to the Tp value of 19.9 min. obtained with the DNA from only 7 viable CFU. These results indicate that even though 2000 dead cells yielded a Tp value of 33.1 min., low numbers of viable cells in the presence of much higher numbers of dead cells still yielded a linear plot for enumerating viable CFU from Tp values. In addition, propidium monoazide (PMA) was found to be ineffective in distinguishing between low numbers of viable and heat killed cells of S. enterica.