Microbial-derived products as potential new antimicrobials.

Due to the continuing global concerns involving antibiotic resistance, there is a need for scientific forums to assess advancements in the development of antimicrobials and their alternatives that might reduce development and spread of antibiotic resistance among bacterial pathogens. The objectives of the 2nd International Symposium on Alternatives to Antibiotics were to highlight promising research results and novel technologies that can provide alternatives to antibiotics for use in animal health and production, assess challenges associated with their authorization and commercialization for use, and provide actionable strategies to support their development. The session on microbial-derived products was directed at presenting novel technologies that included exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials, probiotics development via fecal microbiome transplants among monogastric production animals such as chickens and mining microbial sources such as bacteria or yeast to identify new antimicrobial compounds. Other research has included continuing development of antimicrobial peptides such as newly discovered bacteriocins as alternatives to antibiotics, use of bacteriophages accompanied by development of unique lytic proteins with specific cell-wall binding domains and novel approaches such as microbial-ecology guided discovery of anti-biofilm compounds discovered in marine environments. The symposium was held at the Headquarters of the World Organisation for Animal Health (OIE) in Paris, France during 12-15 December 2016.

Determination of phytase activity in feed: interlaboratory study.

An interlaboratory study was conducted to determine the performance characteristics of a new method for the determination of phytase activity in feed samples. The method is based on the principle that inorganic phosphate is released from the substrate phytate under defined assay conditions and has been validated for its suitability to measure the enzyme activity of various phytase products. Two different experimental designs of the study were applied, allowing for the estimation of the precision of the method under repeatability, intermediate precision and reproducibility conditions, respectively. The relative standard deviation for repeatability (RSDr) ranged from 2.2 to 10.6% and the RSD for reproducibility (RSDR) ranged from 5.4 to 15%. The suitability of the validated method for the intended purpose was demonstrated. The obtained performance profile of the method validated in this study was comparable to that of similar methods that were exclusively validated for one phytase product.

Immunotoxicity of aflatoxin B1: impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression.

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 microg pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-alpha, IL-1beta, IL-6, IFN-gamma) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-gamma and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1.

Determination of selenocysteine and selenomethionine in edible animal tissues by 2D size-exclusion reversed-phase HPLC-ICP MS following carbamidomethylation and proteolytic extraction.

A method was developed for the simultaneous determination of selenomethionine (SeMet) and selenocysteine (SeCys) in meat (chicken and lamb muscles) and different offal tissues (heart, liver, kidney). The analytical procedure was based on the protein extraction with urea under reducing conditions (dithiothreitol), derivatization of SeCys and SeMet by carbamidomethylation with iodoacetamide (IAM) followed by quantitative proteolysis. The mixture of the derivatized Se-amino acids was purified by size-exclusion liquid chromatography (LC) and analysed by ion-paring reversed-phase HPLC-inductively coupled plasma mass spectroscopy (ICP MS). The quantification of SeCys and SeMet was carried out by the method of standard additions. (77)SeMet was used to control the SeMet derivatization efficiency and recovery. The method was validated by the determination of the Se mass balance. The Se-amino acids accounted for 91 +/- 8% of the total selenium (mean of 95 samples of seven tissues analysed over a period of 18 months). The method was applied to the discrimination of the contribution of selenoproteins (containing SeCys) and other Se-containing proteins (containing SeMet) in tissues of animals during supplementation studies (dose-effect and tolerance).

Selective impairment of drug-metabolizing enzymes in pig liver during subchronic dietary exposure to aflatoxin B1.

Consequences of subchronic exposure to aflatoxin B1 (AFB1) on liver monooxygenase and transferase enzymes were compared in control pigs and pigs given 385, 867 or 1,807 microg AFB1/kg of feed for 4 weeks. Animals exposed to the highest dose of toxin developed clinical signs of aflatoxicosis, like liver fibrosis, hepatic dysfunction and decreased weight gain. This group had significantly lower levels of liver cytochrome P450, ethoxyresorufin O-deethylase (EROD) activity, testosterone metabolism, P450 1A and P450 3A protein expression. By comparison, mild degenerative hepatic changes, no hepatic dysfunction but a similar pattern of liver P450 enzymes activity without changes in P450 3A expression were observed in pigs exposed to 867 microg AFB1/kg of feed. Benzphetamine and aminopyrine N-demethylase activities were increased in pigs exposed to 867 or 1,807microg AFB1/kg of feed. Pigs exposed to 385 microg AFB1/kg of feed had low levels of EROD activity and all other biotransformation and clinical parameters remained at control levels. Aniline hydroxylase activity, P450 2C protein expression, UDP-glucuronosyl and glutathione S-transferase activities were unaffected at all doses of AFB1. In conclusion, P450 1A and P450 3A appear to be specific targets of AFB1 even if pig did not display clinical sign of liver toxicosis.

Chemical and conformational study of the interactions involved in mycotoxin complexation with beta-D-glucans.

In a previous paper we reported that beta-D-glucans isolated from Saccharomyces cerevisiae could adsorb zearalenone, reduce its bioavailability in the digestive tract, and protect animals against its adverse effects. We have now investigated, in vitro, the kinetics of the interaction between other mycotoxins and beta-D-glucans from several sources at three pH values found along the digestive tract (3.0, 6.0, and 8.0). Acid and neutral conditions gave the highest affinity rates for aflatoxins B1 > deoxynivalenol > ochratoxin A and involved both the (1 --> 3)-beta-D-glucans and the (1 --> 6)-beta-D-glucans. Alkaline conditions, owing to their destructuring action on glucans, were favorable only for the adsorption of patulin. Using molecular mechanics, we found that hydroxyl, ketone, and lactone groups are involved in the formation of both hydrogen bonds and van der Waals interactions between aflatoxins B1, deoxynivalenol and patulin, and beta-D-glucans. Differences in the binding capacity of the mycotoxins are due to their specific physical and chemical characteristics.

Analysis of the selenium species distribution in cow blood by size exclusion liquid chromatography-inductively coupled plasma collision cell mass spectrometry (SEC-ICPccMS).

A method for performing rapid semiquantitative screening of the distribution of Se species in the blood of cows fed with a diet enriched in selenized yeast was optimized. The method was based on direct injection of a blood sample onto a high resolution size exclusion chromatographic column and fractionation of the selenium species. Selenium was detected on-line by ICP-MS with a collision cell. The concentrations of selenized haemoglobin and free selenomethionine were estimated using the chromatogram. The method was applied to a study involving 15 control and 15 treated dairy cows at four different supplementation time points. The increase in the selenomethionine and selenized haemoglobin was a linear function of the total selenium concentration. A threshold value of 600 ng ml(-1) of total Se was established beyond which selenomethionine could not be incorporated into the protein. No inorganic selenium was found to be present. The total selenium in cow blood correlated well with that in milk. The selenium supplementation did not change the protein distribution profiles for other essential elements (Cu, Fe, Mn, Zn).

Comprehensive conformational study of key interactions involved in zearalenone complexation with beta-D-glucans.

The beta-D-glucans from the cell wall of Saccharomyces cerevisiae have shown in vitro affinity for zearalenone. For this reason, their utilization as dietary adsorbent, to reduce the bioavailability of zearalenone, is of practical interest. Our study used powerful devices to elucidate the spatial conformation and molecular sites of interaction between ZEN and beta-D-glucans. In this respect, 1H NMR spectroscopy implicated the hydroxyl groups of the phenol moiety of zearalenone in the complexation by laminarin, a pure beta-(1,3)-D-glucan. X-ray diffraction determined that laminarin displays the conformation of a single-helix with six beta-D-glucopyranose residues per turn. At this stage, molecular modeling was useful to locate the interaction sites and to propose highly probable complexes of zearalenone with laminarin fragment. Interestingly, the beta-(1,3)-D-glucan chain favors a very stable intra-helical association with zearalenone, nicely stabilized by beta-(1,6)-D-glucans side chains. Both hydrogen bonds and van der Waals interactions were precisely identified in the complex and could thus be proposed as driving interactions to monitor the association between the two molecules.

Validation of the official control method based on Polymerase Chain Reaction (PCR) for identification of authorised probiotic yeast in animal feed.

Council Directive 70/524/EEC regulates the application of probiotic (microorganisms) additives in feeding stuffs. In the present study a method for the differentiation and strain identification of authorised probiotic Saccharomyces cereviseae strains in feeding stuffs by Polymerase Chain Reaction (PCR) was validated. Four different samples of animal feeding stuffs containing yeast at levels between 10(5) to 10(7) CFU/g were examined. Samples were enumerated on chloramphenicol glucose yeast extract agar and colonies were selected from these plates for DNA extraction and subsequent analysis. The PCR method using delta sequence primers produced an 'amplified sequence polymorphism' characteristic for the test strain. Feeds supplemented with one of four probiotic yeast strains each were analysed by seven of nine invited laboratories. All laboratories returned valid results with the exception of one laboratory that had insufficiently separated bands on the gel. The method had a good reproducibility for probiotic yeast isolates from feed of all four authorised probiotic yeast strains (APYS) CBS 493.94, APYS CNCM 1-1079, APYS CNCM 1-1077, APYS NCYC SC47 and of a commercially available yeast reference strain, NCYC 81. The PCR method is to be considered by CEN and ISO as official control method for identification of authorised probiotic Saccharomyces cerevisiae strains from feeding stuffs.

Alkali extraction of beta-d-glucans from Saccharomyces cerevisiae cell wall and study of their adsorptive properties toward zearalenone.

The isolated cell wall of Saccharomyces cerevisiae has some capacity to adsorb zearalenone (affinity near 30%) and reduce the bioavailability of toxins in the digestive tract. The adsorption process was quantified in vitro, and the data obtained when plotted with Hill's equation indicated a cooperative process. The model showed that the adsorption capacity was related to the yeast cell wall composition. This work focused on the role of various beta-d-glucan types in the efficacy of zearalenone adsorption by yeast cell wall and sought to elucidate some of the adsorption mechanisms. Zearalenone was mixed at 37 degrees C with a constant quantity of alkali-soluble or alkali-insoluble beta-d-glucans isolated from yeast cell walls, and the amount of adsorbed zearalenone was measured. Given that the alkali solubility of beta-d-glucans is a determining factor for their three-dimensional conformation and that the alkali-insoluble fraction had a greater affinity (up to 50%) than the alkali-soluble fraction ( approximately 16%), it was concluded that the three-dimensional structure strongly influences the adsorption process. The alkali insolubility of beta-d-glucans led to the formation of single and/or triple helices, which have been identified as the most favorable structures for zearalenone adsorption efficacy. The beta(1,3)-d-glucan and beta(1,6)-d-glucan compositions of the two alkali-extracted fractions and their involvement in the adsorption process are discussed.

A novel technique to evaluate interactions between Saccharomyces cerevisiae cell wall and mycotoxins: application to zearalenone.

Three models based on sigmoidal plotting were tested for their ability to describe zearalenone adsorption on Saccharomyces cerevisiae cell walls in vitro. All three models closely fitted the experimental data, but Hill's equation gave the most accurate parameters, and provided information on the physical and chemical mechanisms involved in the adsorption of mycotoxin on yeast cell walls.

Validation of an official control method for enumeration of authorised probiotic yeast in animal feed.

An official control method in the framework of Council Directive 70/524/EEC for probiotic yeast used as feed additives was validated in a collaborative study by twenty laboratories in 12 European Countries. A pour plate method following ISO 7954 using chloramphenicol glucose yeast extract (CGYE) and a plate count method using CHROMagar Candida were used. Precision data in terms of repeatability (r) and reproducibility (R) of the method using different feeding stuffs and three inoculation levels were determined. Yeast was present in the samples in mixtures with other probiotic feed additives at a lower, a higher concentration or not present. The enumeration of yeast on CGYE agar showed for the lower and higher concentration a RSD(r) of 2.4-4.9% and a RSD(R) of 7.7-8%, respectively and was preferred by the majority of labs. CHROMagar Candida had a RSD(r) of 1.9-2.8% and a RSD(R) of 1.9-5.9%. For routine analysis the use of the pour plate technique is recommended. CHROMagar Candida can be used for confirmation of the species Saccharomyces cerevisiae. The methods are not recommended for mineral feeds. The results from this study are intended for consideration for adoption as CEN and ISO standards.