SCFAs improve disease resistance via modulate gut microbiota, enhance immune response and increase antioxidative capacity in the host.

To evaluate the effects of dietary short chain fatty acids (SCFAs) on the intestinal health and innate immunity in crucian carp, a six-week feeding trial was carried out with following treatments: basal diet (BD), basal diet supplementation with 1% sodium acetate (BDSA), basal diet supplementation with 1% sodium propionate (BDSP) and basal diet supplementation with 1% sodium butyrate (BDSB). The results showed dietary BDSA, BDSP and BDSB could protect the host against oxidative stress by improving the activity of certain antioxidative enzymes (T-SOD, GSH-Px and CAT). Additionally, dietary SCFAs could enhance mucosal and humoral immune responses by improving certain innate immune parameters in serum and skin mucus productions (IgM, ACH50 and T-SOD). Furthermore, dietary BDSA and BDSP could up-regulate the expression of immune related genes (TNF-α, TGF-ß and IL-8) and tight junction protein genes (occludin and ZO-1). Dietary BDSB could also elevate the expression of IL-8, TGF-ß, ZO-1 and Occludin in the midgut. Although dietary differences of SCFAs didn't alter the α-diversity of the intestinal flora, they altered the core microbiota. Finally, the challenge trial showed that dietary basal diet supplementation with SCFAs could protect zebrafish against Aeromonas hydrophila. These results suggest that dietary SCFAs could improve innate immunity, modulate gut microbiota and increase disease resistance in the host, which indicated the potential of SCFAs as immunostimulants in aquaculture.

Spirulina maxima Derived Pectin Nanoparticles Enhance the Immunomodulation, Stress Tolerance, and Wound Healing in Zebrafish.

In this study, Spirulina maxima derived pectin nanoparticles (SmPNPs) were synthesized and multiple biological effects were investigated using in vitro and in vivo models. SmPNPs were not toxic to Raw 264.7 cells and zebrafish embryos up to 1 mg/mL and 200 µg/mL, respectively. SmPNPs upregulated Il 10, Cat, Sod 2, Def 1, Def 2, and Muc 1 in Raw 264.7 cells and tlr2, tlr4b, tlr5b, il1ß, tnfα, cxcl8a, cxcl18b, ccl34a.4, ccl34b.4, muc5.1, muc5.2, muc5.3, hamp, cstd, hsp70, cat, and sod1 in the larvae and adult zebrafish, suggesting immunomodulatory activity. Exposure of larvae to SmPNPs followed by challenge with pathogenic bacterium Aeromonas hydrophila resulted a two-fold reduction of reactive oxygen species, indicating reduced oxidative stress compared to that in the control group. The cumulative percent survival of larvae exposed to SmPNPs (50 µg/mL) and adults fed diet supplemented with SmPNPs (4%) was 53.3% and 76.7%, respectively. Topical application of SmPNPs on adult zebrafish showed a higher wound healing percentage (48.9%) compared to that in the vehicle treated group (38.8%). Upregulated wound healing markers (tgfß1, timp2b, mmp9, tnfα, il1ß,ccl34a.4, and ccl34b.4), enhanced wound closure, and restored pigmentation indicated wound healing properties of SmPNPs. Overall, results uncover the multiple bioactivities of SmPNPs, which could be a promising biocompatible candidate for broad range of aquatic and human therapies.

Quantification of Natural Growth of Two Strains of Mycobacterium Marinum for Translational Antituberculosis Drug Development.

The zebrafish infected with Mycobacterium marinum (M. marinum) is an attractive tuberculosis disease model, showing similar pathogenesis to Mycobacterium tuberculosis (M. tuberculosis) infections in humans. To translate pharmacological findings from this disease model to higher vertebrates, a quantitative understanding of the natural growth of M. marinum in comparison to the natural growth of M. tuberculosis is essential. Here, the natural growth of two strains of M. marinum, E11 and MUSA , is studied over an extended period using an established model-based approach, the multistate tuberculosis pharmacometric (MTP) model, for comparison to that of M. tuberculosis. Poikilotherm-derived strain E11 and human-derived strain MUSA were grown undisturbed up to 221 days and viability of cultures (colony forming unit (CFU)/mL) was determined by plating at different time points. Nonlinear mixed effects modeling using the MTP model quantified the bacterial growth, the transfer among fast, slow, and non-multiplying states, and the inoculi. Both strains showed initial logistic growth, reaching a maximum after 20-25 days for E11 and MUSA , respectively, followed by a decrease to a new plateau. Natural growth of both E11 and MUSA was best described with Gompertz growth functions. For E11, the inoculum was best described in the slow-multiplying state, for MUSA in the fast-multiplying state. Natural growth of E11 was most similar to that of M. tuberculosis, whereas MUSA showed more aggressive growth behavior. Characterization of natural growth of M. marinum and quantitative comparison with M. tuberculosis brings the zebrafish tuberculosis disease model closer to the quantitative translational pipeline of antituberculosis drug development.

Modulatory effects of Amukkara Choornam on Candida albicans biofilm: in vitro and in vivo study.

In the present study, Amukkara Chooram (AC) a well known herbal medicine was investigated for their antibiofilm efficacy against biofilm of Candida albicans. The biofilm inhibitory concentration of 20 µg/mL of AC showed promising effect by inhibiting the biofilm upto 60%. Morphogenic transition state of C. albicans from yeast cells to hyphal transition was prevented by AC was revealed from light microscopic images. In addition, the inhibition of yeast hyphae was examined in the induction medium supplemented with AC. Consequently, atomic force microscope (AFM) also documented the morphological changes observed during the transition state of C. albicans in the presence and absence of AC. Furthermore, scanning electron microscope (SEM) and confocal laser scanning microscopic (CLSM) images showed reduction in the biomass and thickness of the mature biofilm of C. albicans. In vivo investigation of C. albicans with zebrafish infection model presented the clearance of biofilm from the epithelium of the intestinal tissues. Later, the histological changes in liver and kidney due to C. albicans infection open up that treatment with AC was able to significantly rejuvenate the tissues. Altogether, the study presents AC as potent antibiofilm agent with potential ability as alternative medicine to treat C. albicans biofilm mediated infections.

Dietary Supplementation With ω6 LC-PUFA-Rich Algae Modulates Zebrafish Immune Function and Improves Resistance to Streptococcal Infection.

Arachidonic acid (ARA, 20:4n-6) and dihomo-γ-linolenic acid (DGLA, 20:3n-6) are omega-6 long-chain polyunsaturated fatty acids (LC-PUFA), which are key precursors for lipid mediators of the immune system and inflammatory response. The microalga Lobosphaera incisa (WT) and its Δ5-desaturase mutant P127 (MUT) are unique photosynthetic sources for ARA and DGLA, respectively. This study explores the effect of dietary supplementation with L. incisa and P127 biomass on tissue fatty acid composition, immune function, and disease resistance in zebrafish (Danio rerio). The broken microalgal biomass was added to commercial fish feed at 7.5 and 15% (w/w), providing 21.8 mg/g feed ARA for the WT-supplemented group and 13.6 mg/g feed DGLA for the MUT-supplemented group at the 15% inclusion levels. An unsupplemented group was used as the control. After 1 month of feeding, fish were challenged with Streptococcus iniae. Fish were sampled before the challenge and 1 week after the challenge for various analyses. Tissue ARA and DGLA levels significantly increased in the liver, corresponding to microalgal supplementation levels. The elevated expression of specific immune-related genes was evident in the kidneys in all treatment groups after 1 month of feeding, including genes related to eicosanoid synthesis, lysozyme, and NF-κB. In the liver, microalgal supplementation led to the upregulation of genes related to immune function and antioxidant defense while the expression of examined genes involved in ARA metabolism was downregulated. Importantly, fish fed with 15% of both WT- and MUT-supplemented feed showed significantly (p < 0.05) higher survival percentages (78 and 68%, respectively, as compared to only 46% in the control group). The elevated expression of genes related to inflammatory and immune responses was evident post-challenge. Collectively, the results of the current study demonstrate the potential of microalgae-derived dietary ARA and DGLA in improving immune competence and resistance to bacterial infection in zebrafish as a model organism.

Powder of the White Bottom Mushroom, Agaricus bisporus (Agaricomycetes), Improved Immunomodulatory and Health-Promoting Effects of Lactobacillus casei in Zebrafish (Danio rerio).

This study was conducted to investigate the effects of Lactobacillus casei and Agaricus bisporus administration, alone or in combination, on skin mucus immune parameters and the expression of selected genes related to growth, appetite, mucosal immunity, and antioxidant enzymes in zebrafish (Danio rerio). Zebrafish (n = 600) were randomly stocked in 12 aquariums and assigned to 1 of 4 treatments, which were repeated in triplicate. During a 60-day feeding trial, fish were fed experimental diets: (1) basal diet (control group); (2) basal diet + L. casei (107 colony-forming units/g; Pro group); (3) basal diet + 1% A. bisporus [mushroom powder [MP] group); and (4) basal diet + 1% A. bisporus + L. casei (107 colony-forming units/g) (MP + Pro group). The results demonstrated that oral administration of A. bisporus + L. casei significantly (P < 0.05) upregulated the expression of growth-related genes (gh and igf1). Similar results were observed for expression of mucosal immune-related genes (tnf-alpha, lyz, and il1b) and for antioxidant-related genes (sod, cat) in zebrafish fed A. bisporus + L. casei (P < 0.05). Also, nonspecific skin mucus immune parameters in the MP + Pro group were significantly (P < 0.05) higher than those in the MP, Pro, and control groups. These results also demonstrate the potential of combined administration of A. bisporus and L. casei as a beneficial feed additive in aquaculture.

Plant nutraceuticals (Quercetrin and Afzelin) capped silver nanoparticles exert potent antibiofilm effect against food borne pathogen Salmonella enterica serovar Typhi and curtail planktonic growth in zebrafish infection model.

Purified plant nutraceuticals afzelin and quercetrin from an edible plant- Crotolaria tetragona was employed for the fabrication of silver nanoparticles (AgNPs) by a sunlight mediated process. From among a panel of strains tested, AgNPs displayed potent bacteriostatic and bactericidal effect against P. aeruginosa and S. Typhi. Time kill studies revealed green synthesized AgNPs displayed comparable bactericidal effect with chemically synthesized AgNPs against S. Typhi. Antibiofilm potential of AgNPs showed that they were highly effective at sub MIC concentrations in causing 50% biofilm inhibition against food borne pathogen S. Typhi implying that antibiofilm effect is independent of antibacterial effect, which was evidenced by fluorescent imaging and SEM imaging. Mechanistic studies revealed that reduced cell surface hydrophobicity, decreased surface adherence, loss of membrane potential contributed to antibiofilm potential of afzelin/quercetrin AgNPs. Green synthesized afzelin/quercetrin AgNPs were also relatively less toxic and more effective in curtailing bioburden of S. Typhi in infected zebrafish by > 3 log fold. Ability of sunlight reduced afzelin/quercetrin NPs to mitigate planktonic mode of growth in vitro and in vivo and curtail biofilm formation of S. Typhi in vitro demonstrates its potential to curtail food borne pathogen in planktonic and biofilm mode of growth.

Effect of microalgae on intestinal inflammation triggered by soybean meal and bacterial infection in zebrafish.

Soybean meal has been used in many commercial diets for farm fish; despite this component inducing intestinal inflammation. On the other hand, microalgae have increasingly been used as dietary supplements in fish feed. Nevertheless, the vast quantity of microalgae species means that many remain under- or unstudied, thus limiting wide scale commercial application. In this work, we evaluated the effects to zebrafish (Danio rerio) of including Tetraselmis sp (Ts); Phaeodactylum tricornutum (Pt); Chlorella sp (Ch); Nannochloropsis oculata (No); or Nannochloropsis gaditana (Ng) as additives in a soybean meal-based diet on intestinal inflammation and survival after Edwardsiella tarda infection. In larvae fed a soybean meal diet supplemented with Ts, Pt, Ch, or Ng, the quantity of neutrophils present in the intestine drastically decreased as compared to larvae fed only the soybean meal diet. Likewise, Ts or Ch supplements in soybean meal or fishmeal increased zebrafish survival by more than 20% after being challenged. In the case of Ts, the observed effect correlated with an increased number of neutrophils present at the infection site. These results suggest that the inclusion of Ts or Ch in fish diets could allow the use of SBM and at the same time improve performance against pathogen.

In vitro and in vivo antifungal efficacy of plant based lawsone against Fusarium oxysporum species complex.

Fusarium oxysporum is an ascomycete facultative fungus which generally affects to plants. However, it is recently known as a serious emerging opportunistic pathogen of human and other animals. F. oxysporum shows broad resistance to commonly used antifungal agents and therefore development of alternative therapeutic agents is required. In this study, we investigated the antifungal efficacy of plant based natural lawsone against pathogenic F. oxysporum. Antifungal susceptibility test determined the concentration dependent growth inhibition of lawsone against F. oxysporum with minimum inhibitory concentration (MIC) at 100µg/mL. Ultra-structural analysis indicates the prominent damage on cell wall of the mycelium after lawsone treatment, and suggests that it could increase the membrane permeability and disintegration of cells leading to cellular death. Propidium iodide (PI) uptake assay results showed the higher level of cell death in lawsone treated F. oxysporum which further confirms the loss of plasma membrane integrity. Also, detection of reactive oxygen species (ROS) using DCFH-DA has clearly indicated that lawsone (100µg/mL) can induce the ROS level in the filaments of F. oxysporum. MTT assay results showed the loss of viability and germination capacity of F. oxysporum spores by lawsone in concentration dependent manner. Moreover, lawsone treatment induced the mRNA expression of two autophagy related genes (ATG1 and ATG8) indicating that lawsone may activate the autophagy related pathways in F. oxysporum due to the oxidative stress generated by ROS. F. oxysporum infected zebrafish has recovered after lawsone therapy as a topical treatment suggesting that lawsone is a potential natural antifusariosis agent.

The Growth-Promoting Effect of Dietary Nucleotides in Fish Is Associated with an Intestinal Microbiota-Mediated Reduction in Energy Expenditure.

Background: Nucleotides have been used as functional nutrients to improve the growth and health of animals, including fish. The mechanism involved in the growth-promotion effect of nucleotides is still unclear.Objective: We investigated the bioenergetic mechanism underlying the growth-promotion effect of nucleotides in zebrafish and the associated roles played by the intestinal microbiota.Methods: Larval zebrafish were fed a control or a 0.1% mixed nucleotides-supplemented diet for 2 wk. Standard metabolic rate, the minimal rate of energy expenditure by animals at rest, was evaluated by oxygen consumption with the use of a respirometer. The expressions of fasting-induced adipose factor (Fiaf), inflammatory cytokines, and genes involved in fatty acid (FA) oxidation were tested by quantitative reverse transcriptase-polymerase chain reaction. The intestinal microbiota from the nucleotide-fed fish (NT fish) or control fish was transferred to 3-d postfertilization germ-free zebrafish in which oxygen consumption and expression of cytokines and fiaf were evaluated.Results: Compared with controls, nucleotide supplementation at 0.1% increased the weight and energy gains of zebrafish by 10% and 25%, respectively (P < 0.01). Standard metabolic rate was 28% lower in NT fish than in controls (P < 0.001). Nucleotide supplementation downregulated the inflammatory tone in the head kidney of the fish. Moreover, NT fish had a 51% lower intestinal expression of fiaf than did controls (P < 0.05), which was consistent with decreased expression of key genes involved in FA oxidation [carnitine:palmitoyl transferase 1a (cpt1a) and medium-chain acyl coenzyme A dehydrogenase (mcad)] in liver and muscle. Germ-free zebrafish colonized with microbiota from NT fish had a 25% lower standard metabolic rate than did those colonized by control microbiota (P < 0.01), whereas direct nucleotide feeding of germ-free zebrafish did not affect standard metabolic rate relative to germ-free controls that were not fed nucleotides. Furthermore, germ-free zebrafish colonized with nucleotide microbiota exhibited downregulated inflammatory tone and 33% lower fiaf expression compared with their control microbiota-colonized counterparts.Conclusions: The growth-promoting effect of dietary nucleotides in zebrafish involves 2 intestinal microbiota-mediated mechanisms that result in reduced standard metabolic rate: 1) lower inflammatory tone and 2) reduced FA oxidation associated with increased microbial suppression of intestinal fiaf.

Screening of anti-mycobacterial compounds in a naturally infected zebrafish larvae model.

OBJECTIVES: Mycobacterium tuberculosis is a deadly human pathogen that causes the lung disease TB. M. tuberculosis latently infects a third of the world's population, resulting in ∼1.5 million deaths per year. Due to the difficulties and expense of carrying out animal drug trials using M. tuberculosis and rodents, infections of the zebrafish Danio rerio with Mycobacterium marinum have become a useful surrogate. However, the infection methods described to date require specialized equipment and a high level of operator expertise. METHODS: We investigated whether zebrafish larvae could be naturally infected with bioluminescently labelled M. marinum by immersion, and whether infected larvae could be used for rapid screening of anti-mycobacterial compounds using bioluminescence. We used rifampicin and a variety of nitroimidazole-based next-generation and experimental anti-mycobacterial drugs, selected for their wide range of potencies against M. tuberculosis, to validate this model for anti-mycobacterial drug discovery. RESULTS: We observed that five of the six treatments (rifampicin, pretomanid, delamanid, SN30488 and SN30527) significantly reduced the bioluminescent signal from M. marinum within naturally infected zebrafish larvae. Importantly, these same five treatments also retarded the growth of M. tuberculosis in vitro. In contrast, only three of the six treatments tested (rifampicin, delamanid and SN30527) retarded the growth of M. marinum in vitro. CONCLUSIONS: We have demonstrated that zebrafish larvae naturally infected with bioluminescent M. marinum M can be used for the rapid screening of anti-mycobacterial compounds with readily available equipment and limited expertise. The result is an assay that can be carried out by a wide variety of laboratories for minimal cost and without high levels of zebrafish expertise.

Thymol and Carvacrol Affect Hybrid Tilapia through the Combination of Direct Stimulation and an Intestinal Microbiota-Mediated Effect: Insights from a Germ-Free Zebrafish Model.

BACKGROUND: Essential oils (EOs) are commonly used as animal feed additives. Information is lacking on the mechanisms driving the beneficial effects of EOs in animals, especially the role played by the intestinal microbiota of the host. OBJECTIVE: The purpose of this study was to clarify the relative contribution of direct effects of EOs on the physiology and immune system of tilapia and indirect effects mediated by the intestinal microbiota by using a germ-free zebrafish model. METHODS: Juvenile hybrid tilapia were fed a control diet or 1 of 4 treatment diets containing 60-800 mg Next Enhance 150 (NE) (an EO product containing equal levels of thymol and carvacrol)/kg for 6 wk. The key humoral and cellular innate immune parameters were evaluated after the feeding period. In another experiment, the gut microbiota of tilapia fed a control or an NE diet (200 mg/kg) for 2 wk were transferred to 3-d postfertilization (dpf) germ-free (GF) zebrafish, and the expression of genes involved in innate immunity and tight junctions was evaluated in zebrafish at 6 dpf. Lastly, NE was directly applied to 3-dpf GF zebrafish at 3 doses ranging from 0.2 to 20 mg/L, and the direct effect of NE on zebrafish was evaluated after 1 and 3 d. RESULTS: NE supplementation at 200 mg/kg enhanced phagocytosis activity of head kidney macrophages (×1.36) (P < 0.05) and plasma lysozyme activity (×1.69) of tilapia compared with the control (P < 0.001), indicating an immunostimulatory effect. Compared with those colonized with control microbiota, GF zebrafish colonized with NE microbiota showed attenuated induction of immune response marker genes serum amyloid a (Saa; ×0.62), interleukin 1ß (Il1ß; ×0.29), and interleukin 8 (Il8; ×0.62) (P < 0.05). NE treatment of GF zebrafish at 2 and 20 mg/L for 1 d upregulated the expression of Il1ß (×2.44) and Claudin1 (×1.38), respectively (P < 0.05), whereas at day 3 the expression of Occludin2 was higher (×3.30) in the 0.2-mg NE/L group compared with the GF control (P < 0.05). CONCLUSION: NE may affect the immunity of tilapia through a combination of factors, i.e., primarily through a direct effect on host tissue (immune-stimulating) but also an indirect effect mediated by microbial changes (immune-relieving).

Establishment and optimization of a high throughput setup to study Staphylococcus epidermidis and Mycobacterium marinum infection as a model for drug discovery.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

In vivo assessment of drug efficacy against Mycobacterium abscessus using the embryonic zebrafish test system.

Mycobacterium abscessus is responsible for a wide spectrum of clinical syndromes and is one of the most intrinsically drug-resistant mycobacterial species. Recent evaluation of the in vivo therapeutic efficacy of the few potentially active antibiotics against M. abscessus was essentially performed using immunocompromised mice. Herein, we assessed the feasibility and sensitivity of fluorescence imaging for monitoring the in vivo activity of drugs against acute M. abscessus infection using zebrafish embryos. A protocol was developed where clarithromycin and imipenem were directly added to water containing fluorescent M. abscessus-infected embryos in a 96-well plate format. The status of the infection with increasing drug concentrations was visualized on a spatiotemporal level. Drug efficacy was assessed quantitatively by measuring the index of protection, the bacterial burden (CFU), and the number of abscesses through fluorescence measurements. Both drugs were active in infected embryos and were capable of significantly increasing embryo survival in a dose-dependent manner. Protection from bacterial killing correlated with restricted mycobacterial growth in the drug-treated larvae and with reduced pathophysiological symptoms, such as the number of abscesses within the brain. In conclusion, we present here a new and efficient method for testing and compare the in vivo activity of two clinically relevant drugs based on a fluorescent reporter strain in zebrafish embryos. This approach could be used for rapid determination of the in vivo drug susceptibility profile of clinical isolates and to assess the preclinical efficacy of new compounds against M. abscessus.

Probiotic supplementation promotes calcification in Danio rerio larvae: a molecular study.

A growing number of studies have been showing that dietary probiotics can exert beneficial health effects in both humans and animals. We previously demonstrated that dietary supplementation with Lactobacillus rhamnosus - a component of the human gut microflora - enhances reproduction, larval development, and the biomineralization process in Danio rerio (zebrafish). The aim of this study was to identify the pathways affected by L. rhamnosus during zebrafish larval development. Our morphological and histochemical findings show that L. rhamnosus accelerates bone deposition through stimulation of the expression of key genes involved in ossification, e.g. runt-related transcription factor 2 (runx2), Sp7 transcription factor (sp7), matrix Gla protein (mgp), and bone gamma-carboxyglutamate (gla) protein (bglap) as well as through inhibition of sclerostin (sost), a bone formation inhibitor. Western blot analysis of mitogen-activated protein kinase 1 and 3-(Mapk1 and Mapk3), which are involved in osteoblast and osteocyte differentiation, documented an increase in Mapk1 16 days post fertilization (dpf) and of Mapk3 23 dpf in individuals receiving L. rhamnosus supplementation. Interestingly, a reduction of sost detected in the same individuals suggests that the probiotic may help treat bone disorders.

Preliminary evidence of the contribution of the intestinal microflora to biotin supply in zebrafish Danio rerio (Hamilton-Buchanan).

A study was conducted to preliminarily assess the contribution of the intestinal microflora to biotin supply in zebrafish. Biotin and avidin were added to three isonitrogenous and isocaloric purified diets to provide molar avidin: biotin ratios of 0:0 (basal diet), 0:1 (biotin-supplemented diet), and 120:0. Another diet was made by supplementing the antibiotic succinylsulfathiazole (1%, wt/wt) to the basal diet. A fifth diet was the Zeigler commercial diet for zebrafish. Each diet was fed to a triplicate group of fish (mean initial mass 0.266 g) for 8 weeks. The condition factor, feed conversion ratio (FCR), percentage weight gain, and survival were similar in fish groups fed the commercial and the biotin-supplemented diets, but energy conversion efficiency and whole-body biotin content were highest in the fish fed the commercial diet (p<0.05). Reduced growth and survival, and increased FCR were noted in fish fed basal diet compared with those fed biotin-supplemented diet. The supplementation of avidin in diet led to lower survival and condition factor, and higher FCR than that observed with basal diet. Intestinal microbial synthesis is assumed to be a significant source of biotin to the zebrafish, as fish fed the antibiotic-supplemented diet showed the lowest growth, health condition, and feed utilization.

MMAR_2770, a new enzyme involved in biotin biosynthesis, is essential for the growth of Mycobacterium marinum in macrophages and zebrafish.

Biotin, which functions as an essential cofactor for certain carboxylases and decarboxylases, is synthesized by a multistep pathway in microorganisms and plants. Biotin biosynthesis has not been studied in detail in mycobacteria. In this study, we isolated a mutant of Mycobacterium marinum in which MMAR_2770, a previously uncharacterized gene encoding a predicted short-chain dehydrogenase/reductase, was inactivated. We found that this mutant is a biotin auxotroph that cannot grow in a minimal medium (Sauton) unless biotin is supplemented. Complementation of the mutant with an intact MMAR_2770 or its homolog Rv1882c of Mycobacterium tuberculosis restored the growth of the mutant, suggesting that MMAR_2770 is involved in biotin biosynthesis. We further showed that the mutant was unable to grow in cultured macrophages and was attenuated in zebrafish. Taken together, our results demonstrate that biotin biosynthesis is essential for the growth of mycobacteria in vitro and in vivo and have provided validation for targeting biotin biosynthetic enzymes for antimycobacterial drug development. The potential role of MMAR_2770 in mycobacterial biotin biosynthesis is discussed.

Grape seed extract inhibits the growth and pathogenicity of Staphylococcus aureus by interfering with dihydrofolate reductase activity and folate-mediated one-carbon metabolism.

Staphylococcus aureus (S. aureus) is one of the most common pathogens that causes infectious and foodborne diseases worldwide. Searching for drug and chemical compounds against this bacterium is still in demand. We found that grape seed extract (GSE), a natural food product rich in polyphenols, inhibited the dihydrofolate reductase activity and growth of S. aureus. In addition, the intracellular content of tetrahydrofolate (THF), the major folate species identified in S. aureus, was significantly decreased when GSE was present in medium. The GSE-induced growth inhibition was reversed by adding, THF, 5,10-methylenetetrahydrofolate or methionine to the medium. The differential rescuing effects elicited by thymidine and methionine indicated that GSE-induced perturbation in folate-mediated one-carbon metabolism has more profound impact on methionine cycle than on thymidine monophosphate (TMP) synthesis. Significantly reduced inflammatory responses and mortality were observed in zebrafish infected with S. aureus pre-incubated with GSE. We conclude that GSE might serve as an effective natural alternative for the control of food poisoning caused by S. aureus with proper safety measure.

Transgenic zebrafish eggs containing bactericidal peptide is a novel food supplement enhancing resistance to pathogenic infection of fish.

Zebrafish (Danio rerio) was used as a bioreactor to produce bovine lactoferricin (LFB), which has wide-ranging antimicrobial activity. We constructed an expression plasmid in which LFB was fused with green fluorescent protein (GFP) and driven by zebrafish beta-actin promoter. After microinjection, six transgenic founders were screened on the basis of GFP appearance. Among them, a stable ZBL-5 line was selected by the ubiquitous and strong expression of GFP. Using PCR and Western blot analysis, we confirmed that the recombinant LFB-GFP protein was produced by the F2 progeny derived from the ZBL-5 line. The bactericidal agar plate assay proved that the functional domain of LFB was released from the LFB-GFP fusion protein, resulting in strong bactericidal activity against Escherichia coli, Edwardsiella tarda and Aeromonas hydrophila. Furthermore, adult zebrafish were given one feeding of fifty 72-hpf transgenic embryos. The treated fish were then immersed in freshwater containing 1 x 10(5) CFU ml(-1)E. tarda for 7 days. The survival rate of the treated zebrafish was significantly higher than that of fish fed with fifty wild-type embryos (75 +/- 12.5% versus 4 +/- 7.2%). This line of evidence suggested that pathogen resistance can be enhanced by using transgenic embryos containing LFB-GFP as a food supplement for fish, while, at the same time, reducing the demand of chemical antibiotics.

Fishing for new antimicrobials.

The discovery of antibiotics and other antimicrobial agents in the 1930s is arguably the most significant therapeutic advance in medical history. Penicillin and the sulfa drugs touched off the search for and discovery of countless derivative compounds and several new antibiotic classes. However, the pace of discovery has slowed down, and there is growing appreciation that much of the low-lying fruit accessible to traditional methods of antimicrobial discovery has been harvested. Combating emerging drug-resistant strains of infectious agents may require the adoption of fresh approaches to drug target validation, small-molecule discovery and safety assessment. The recent development of several infectious disease models in zebrafish raises the possibility of a new paradigm in antimicrobial discovery.