Summary
The biological detoxification of mycotoxins, including deoxynivalenol (DON), represents a very promising approach to address the challenging problem of cereal grain contamination. The recent ...discovery of Devosia mutans 17‐2‐E‐8 (Devosia spp. 17‐2‐E‐8), a bacterial isolate capable of transforming DON to the non‐toxic stereoisomer 3‐epi‐deoxynivalenol, along with earlier reports of bacterial species capable of oxidizing DON to 3‐keto‐DON, has generated interest in the possible mechanism and enzyme(s) involved. An understanding of these details could pave the way for novel strategies to manage this widely present toxin. It was previously shown that DON epimerization proceeds through a two‐step biocatalysis. Significantly, this report describes the identification of the first enzymatic step in this pathway. The enzyme, a dehydrogenase responsible for the selective oxidation of DON at the C3 position, was shown to readily convert DON to 3‐keto‐DON, a less toxic intermediate in the DON epimerization pathway. Furthermore, this study provides insights into the PQQ dependence of the enzyme. This enzyme may be part of a feasible strategy for DON mitigation within the near future.
This report describes the discovery of a dehydrogenase responsible for the selective oxidation of mycotoxin deoxynivalenol (DON) at the C3 position. The enzyme was shown to readily convert DON to 3‐keto‐DON, a less toxic intermediate in the DON epimerization pathway. This enzyme may be part of a feasible strategy for DON mitigation within the near future.
Mycotoxins, the secondary metabolites of mycotoxigenic fungi, have been found in almost all agricultural commodities worldwide, causing enormous economic losses in livestock production and severe ...human health problems. Compared to traditional physical adsorption and chemical reactions, interest in biological detoxification methods that are environmentally sound, safe and highly efficient has seen a significant increase in recent years. However, researchers in this field have been facing tremendous unexpected challenges and are eager to find solutions. This review summarizes and assesses the research strategies and methodologies in each phase of the development of microbiological solutions for mycotoxin mitigation. These include screening of functional microbial consortia from natural samples, isolation and identification of single colonies with biotransformation activity, investigation of the physiological characteristics of isolated strains, identification and assessment of the toxicities of biotransformation products, purification of functional enzymes and the application of mycotoxin decontamination to feed/food production. A full understanding and appropriate application of this tool box should be helpful towards the development of novel microbiological solutions on mycotoxin detoxification.
Necrotic enteritis (NE) is a poultry intestinal disease caused by virulent strains of the bacterium Clostridium perfringens (C. perfringens). This anaerobic bacterium produces a wide range of enzymes ...and toxins in the gut which leads to NE development. It is generally accepted by the poultry veterinarians that netB-positive C. perfringens strains are virulent and netB-negative strains do not cause NE. However, NE pathogenesis remains unclear as contradictory results have been reported. The use of experimental in vivo models is a valuable tool to understand the pathogenesis of a disease. In this study, a chicken ligated loop model was used to determine the virulence status of 79 C. perfringens strains from various geographical locations, sources, and genotype profiles. According to our model and based on histologic lesion scoring, 9 C. perfringens strains were classified as commensal, 35 as virulent, and 34 as highly virulent. The virulence of only 1 C. perfringens strain could not be classified as its lesion score was variable (from <10 to >15). In general, NE lesions were more severe in intestinal loops inoculated with netB-positive C. perfringens strains than those inoculated with netB-negative strains. The prevalence of netB among strains classified as commensal, virulent, and highly virulent was 56% (5/9), 54%, (19/35), and 59% (20/34). These results suggest that NetB is not required to cause NE lesions and that other factors are also involved. The classification of the virulence status of C. perfringens strains should not be based solely on the presence or absence of this toxin. Therefore, the use of an in vivo model is essential to distinguish commensal from virulent strains of C. perfringens.
This review summarizes advances in understanding the pathogenesis of necrotic enteritis of chickens caused by netB-positive Clostridium perfringens. The discovery of NetB as the essential toxin ...trigger for the disease was followed by recognition that it forms part of a large plasmid-encoded 42 kb pathogenicity locus (NELoc-1). While the locus is critical for toxin production, it likely has additional functions related to colonization and degradation of the mucus barrier, which are essential both to multiplication and to bringing NetB close to the intestinal epithelium. Two "chitinases" (glycoside hydrolases (GHs)) present on NELoc-1 are predicted to be involved in mucin degradation, as is the large carbohydrate-binding metalloprotease, shown to be involved in mucinase activity in other clostridia. A second pathogenicity locus found in netB-positive C. perfringens, NELoc-2, also encodes a GH likely involved in mucin degradation. Upon reaching a sufficient cell density on the intestinal mucosa, the Agr-like quorum-sensing system is triggered, which in turn up-regulates the VirR/VirS regulon. This regulon includes NetB. Where NetB initiates damage is unresolved, but it may be deep in the intestinal mucosa, rather than superficially. As the disease progresses, C. perfringens line what remains of the intestinal epithelium in large numbers. This likely involves a number of different bacterial adhesins, including additional NELoc-1-encoded bacterial surface proteins, some of which may adhere to epithelial cell ligands exposed by bacterial sialidases. Further studies of the pathogenesis of necrotic enteritis should lead to development of novel ways to control the infection.
Deoxynivalenol (DON) is one of the most common mycotoxins found in cereal grains and grains contaminated with DON can cause health issues for both humans and animals and result in severe economic ...losses. Currently there is no feasible method to remediate affected grains. The development of a biological method for detoxification is becoming increasingly more plausible with the discovery of microbes which can transform DON to a relatively non-toxic stereoisomer, 3-
-DON. Although bacteria capable of detoxifying DON have been known for some time, it is only recently an enzyme responsible was identified. In
17-2-E-8 (
sp. 17-2-E-8) a two-step DON epimerization (Dep) pathway, designated as the Dep system, completes this reaction. DepA was recently identified as the enzyme responsible for the conversion of DON to 3-keto-DON, and in this report, DepB, a NADPH dependent dehydrogenase, is identified as the second and final step in the pathway. DepB readily catalyzes the reduction of 3-keto-DON to 3-
-DON. DepB is shown to be moderately thermostable as it did not lose significant activity after a heat treatment at 55°C and it is amenable to lyophilization. DepB functions at a range of pH-values (5-9) and functions equally well in multiple common buffers. DepB is clearly a NADPH dependent enzyme as it utilizes it much more efficiently than NADH. The discovery of the final step in the Dep pathway may provide a means to finally mitigate the losses from DON contamination in cereal grains through an enzymatic detoxification system. The further development of this system will need to focus on the activity of the Dep enzymes under conditions mimicking industrially relevant conditions to test their functionality for use in areas such as corn milling, fuel ethanol fermentation or directly in animal feed.
This study investigated the effects of encapsulated cinnamaldehyde (CIN) and citral (CIT) alone or in combination (CIN + CIT) on the growth performance and cecal microbiota of nonvaccinated broilers ...and broilers vaccinated against coccidiosis. Vaccinated (1,600) and nonvaccinated (1,600) 0-day-old male Cobb500 broilers were randomly allocated to 5 treatments: basal diet (control) and basal diet supplemented with bacitracin (BAC, 55 ppm), CIN (100 ppm), CIT (100 ppm), and CIN (100 ppm) + CIT (100 ppm). In general, body weight (BW) and feed conversion ratio were significantly improved in birds treated with BAC, CIN, CIT, and CIN + CIT (P < 0.05) but were all decreased in vaccinated birds compared with nonvaccinated birds (P < 0.05). Significant interactions (P < 0.05) between vaccination and treatments for average daily gain during the periods of starter (day 0–9) and BW on day 10 were noted. Broilers receiving vaccines (P < 0.01) or feed supplemented with BAC, CIN, CIT, or CIN + CIT (P < 0.01) showed reductions in mortality rate from day 0 to 28. The incidences of minor coccidiosis were higher (P < 0.05) in vaccinated birds than in nonvaccinated birds. Diet supplementation with BAC or tested encapsulated essential oils showed comparable effects on the coccidiosis incidences. Similar to BAC, CIN and its combination with CIT reduced both incidence and severity of necrotic enteritis (P < 0.05). No treatment effects were observed on the cecal microbiota at the phyla level. At the genus level, significant differences between vaccination and treatment groups were observed for 5 (Lactobacillus, Ruminococcus, Faecalibacterium, Enterococcus, and Clostridium) of 40 detected genera (P < 0.05). The genus Lactobacillus was more abundant in broilers fed with CIT, while Clostridium and Enterococcus were less abundant in broilers fed with CIN, CIT, or CIN + CIT in both the vaccinated and nonvaccinated groups. Results from this study suggested that CIN alone or in combination with CIT in feed could improve chicken growth performance to the level comparable with BAC and alter cecal microbiota composition.
Butyrate can modulate the immune response and energy expenditure of animals and enhance intestinal health. The present study investigated changes in the intestinal microbiota composition and serum ...metabolites of young broilers in response to 3,000 ppm butyrate in the form of butyrate glycerides (BG) via pyrosequencing of bacterial 16S rRNA genes and nuclear magnetic resonance (NMR). The dietary treatment did not affect the alpha diversity of intestinal microbiota, but altered its composition. Thirty-nine key operational taxonomic units (OTUs) in differentiating cecal microbiota community structures between BG treated and untreated chickens were also identified. Bifidobacterium was, in particular, affected by the dietary treatment significantly, showing an increase in not only the abundance (approximately 3 fold, P ≤ 0.05) but also the species diversity. The (NMR)-based analysis revealed an increase in serum concentrations of alanine, low-density and very low-density lipoproteins, and lipids (P ≤ 0.05) by BG. More interestingly, the dietary treatment also boosted (P ≤ 0.05) serum concentrations of bacterial metabolites, including choline, glycerophosphorylcholine, dimethylamine, trimethylamine, trimethylamine-N-oxide, lactate, and succinate. In conclusion, the data suggest the modulation of intestinal microbiota and serum metabolites by BG dietary treatment and potential contribution of intestinal bacteria to lipid metabolism/energy homeostasis in broilers.
Understanding how dietary components alter the healthy baseline colonic microenvironment is important in determining their roles in influencing gut health and gut-associated diseases. Dietary ...flaxseed (FS) has demonstrated anti-colon cancer effects in numerous rodent models, however, exacerbated acute colonic mucosal injury and inflammation in a colitis model. This study investigates whether FS alters critical aspects of gut health in healthy unchallenged mice, which may help explain some of the divergent effects observed following different gut-associated disease challenges. Four-week-old C57Bl/6 male mice were fed an AIN-93G basal diet (BD) or an isocaloric BD+10% ground FS diet for 3 weeks. FS enhanced colon goblet cell density, mucus production, MUC2 mRNA expression, and cecal short chain fatty acid levels, indicative of beneficial intestinal barrier integrity responses. Additionally, FS enhanced colonic regenerating islet-derived protein 3 gamma (RegIIIγ) and reduced MUC1 and resistin-like molecule beta (RELMβ) mRNA expression which may indicate altered responses in regulating microbial defense and injury repair responses. FS diet altered the fecal microbial community structure (16S rRNA gene profiling), including a 20-fold increase in Prevotella spp. and a 30-fold reduction in Akkermansia muciniphila abundance. A 10-fold reduction in A. muciniphila abundance by FS was also demonstrated in the colon tissue-associated microbiota (quantitative PCR). Furthermore, fecal branched chain fatty acids were increased by FS, indicative of increased microbial-derived putrefactive compounds. In conclusion, consumption of a FS-supplemented diet alters the baseline colonic microenvironment of healthy mice which may modify subsequent mucosal microbial defense and injury-repair responses leading to altered susceptibility to different gut-associated diseases.
Deoxynivalenol (DON) is a secondary fungal metabolite that is associated with many adverse toxicological effects in agriculture as well as human/animal nutrition. Bioremediation efforts in recent ...years have led to the discovery of numerous bacterial isolates that can transform DON to less toxic derivatives. Both 3-keto-DON and 3-epi-DON were recently shown to exhibit reduced toxicity, compared to DON, when tested using different cell lines and mammalian models. In the current study, the toxicological assessment of 3-keto-DON and 3-epi-DON using in planta models surprisingly revealed that 3-keto-DON, but not 3-epi-DON, retained its toxicity to a large extent in both duckweeds (Lemna minor L.) and common wheat (Triticum aestivum L.) model systems. RNA-Seq analysis revealed that the exposure of L. minor to 3-keto-DON and DON resulted in substantial transcriptomic changes and similar gene expression profiles, whereas 3-epi-DON did not. These novel findings are pivotal for understanding the environmental burden of the above metabolites as well as informing the development of future transgenic plant applications. Collectively, they emphasize the fundamental need to assess both plant and animal models when evaluating metabolites/host interactions.
Non-typhoidal Salmonella enterica serovars continue to be an important food safety issue worldwide. Cranberry (Vaccinium macrocarpon Ait) fruits possess antimicrobial properties due to their various ...acids and phenolic compounds; however, the underlying mechanism of actions is poorly understood. We evaluated the effects of cranberry extracts on the growth rate of Salmonella enterica serovars Typhimurium, Enteritidis and Heidelberg and on the transcriptomic profile of Salmonella Enteritidis to gain insight into phenotypic and transcriptional changes induced by cranberry extracts on this pathogen. An ethanolic extract from cranberry pomaces (KCOH) and two of its sub-fractions, anthocyanins (CRFa20) and non-anthocyanin polyphenols (CRFp85), were used. The minimum inhibitory (MICs) and bactericidal (MBCs) concentrations of these fractions against tested pathogens were obtained using the broth micro-dilution method according to the Clinical Laboratory Standard Institute's guidelines. Transcriptional profiles of S. Enteritidis grown in cation-adjusted Mueller-Hinton broth supplemented with or without 2 or 4 mg/ml of KCOH were compared by RNASeq to reveal gene modulations serving as markers for biological activity. The MIC and MBC values of KCOH were 8 and 16 mg/mL, respectively, against all tested S. enterica isolates. The MIC value was 4 mg/mL for both CRFa20 and CRFp85 sub-fractions, and a reduced MBC value was obtained for CRFp85 (4 mg/ml). Treatment of S. Enteritidis with KCOH revealed a concentration-dependent transcriptional signature. Compared to the control, 2 mg/ml of KCOH exposure resulted in 89 differentially expressed genes (DEGs), of which 53 and 36 were downregulated and upregulated, respectively. The upregulated genes included those involved in citrate metabolism, enterobactin synthesis and transport, and virulence. Exposure to 4 mg/ml KCOH led to the modulated expression of 376 genes, of which 233 were downregulated and 143 upregulated, which is 4.2 times more DEGs than from exposure to 2 mg/ml KCOH. The downregulated genes were related to flagellar motility, Salmonella Pathogenicity Island-1 (SPI-1), cell wall/membrane biogenesis, and transcription. Moreover, genes involved in energy production and conversion, carbohydrate transport and metabolism, and coenzyme transport and metabolism were upregulated during exposure to 4 mg/ml KCOH. Overall, 57 genes were differentially expressed (48 downregulated and 9 upregulated) in response to both concentrations. Both concentrations of KCOH downregulated expression of hilA, which is a major SPI-1 transcriptional regulator. This study provides information on the response of Salmonella exposed to cranberry extracts, which could be used in the control of this important foodborne pathogen.
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