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Microsoft word - 05-01-05 final report.doc

Manitoba Livestock Manure Management Initiative Project 05-01-05 “Bioactive Compounds in Swine Manure” Composted swine manure was examined for the presence of antibacterial and antifungal activities. While commercial composts show some antibacterial activity composted swine manure demonstrated inhibitory activity against Gram-positive bacteria and fungi. Weaker inhibitory activity against Gram-negative bacteria was also detected in composted swine manure. It is likely that these antimicrobial activities are due to compounds produced by bacteria or fungi present in the composted swine manure. An antifungal compound of possible commercial and agricultural significance is present in composted swine manure. Antibacterial agents of the same significance are also present. Introduction Manure is usually regarded by the public as a problem while farmers regard manure as a valuable commodity, as a fertilizer. Manure and composted manure are extremely complex bacterial ecosystems. Hundreds, probably thousands, of species of bacteria, fungi, viruses and other microscopic organisms live, grow and die in manure and compost. They must all compete with each other for nutrients and space, consume each other or avoid consumption. To successfully compete bacteria and fungi produce bioactive compounds; antibiotics, antifungal and antiviral compounds and compounds with bioactivity against larger organisms. These bioactivities may include anticancer, anti-inflammatory, anti-oxidative and anti-parasite properties; activities which could be beneficial to the human population. Extensive surveys of natural products from diverse living organisms have been undertaken by major pharmaceutical companies over the past 50 years to discover valuable bioactive compounds. The antibiotics are the most notable success of this endeavour. However no reports of discovering antibiotics or antibiotic-producing organisms from manure or compost have been published. We may be overlooking a valuable source of new pharmaceuticals. The most successful pharmaceutical is the antibiotic. Unfortunately many bacteria have become resistant to our existing antibiotics. Most bacterial pathogens demonstrate resistance to one or many antibiotics. Notorious are the methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). These bacteria are significant causes of human disease and mortality. In some cases there are no antibiotics effective at killing the bacteria leaving medicine in a very difficult position. New types of antibiotics are urgently required. Most currently used antibiotics were discovered in the 1950s and 1960s. Major pharmaceutical companies have left the field of antibiotic discovery and development. Only in the past few years have new antibiotics such as Synercid, daptomycin, and linezolid become available. Even these “new” antibiotics were previously discovered and development abandoned or were previously used in non-human therapy. Few other new types of antibiotics are in development. Given the complexity of the microbial ecology of composted manure antibiotics may be produced there which have novel structures and towards which no resistance has developed in bacterial pathogens. There for we undertook a study to determine whether any antibacterial or antifungal agents could be isolated from composted swine manure. Swine manure was obtained from Landmark Feeds/elite Swine. Approximately 40 liters of manure was obtained from a typical hog barn. The manure was allowed to settle overnight and about 20 liters were decanted to increase the solids content. Perlite and sawdust were added to simulate composting. The manure was sampled after receiving it at the University of Winnipeg and again after 6 weeks and 18 weeks. Samples were frozen at -20 oC. While the composting was in progress we obtained 5 commercial composts to check for the presence of bioactive compounds which may be present in all composts and not specific to swine manure. These composts included commercial steer manure compost, sheep manure compost, two brands of mushroom compost and a “farm” compost. Composts were oven-dried (80oC) to determine gravimetric water content. Larger samples were then dried. A 100 g dry-weight sample of each compost was extracted overnight with ethyl acetate. The solvent was decanted, filtered and evaporated to give an extract. The compost samples were then extracted with hexane overnight. The hexane was decanted, filtered and evaporated to give another extract. Each extract was dissolved in 2 mL of solvent. To test for antibacterial and antifungal activity 50 μL of each extract was applied to paper discs and the solvent was allowed to evaporate. The discs were then applied to the surface of Petri plates inoculated with Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa and the yeast Candida albicans. Plates were incubated overnight and zones of growth inhibition around each disc were recorded. Mueller-Hinton agar was used for the growth of bacteria and Sabourand agar for the growth of yeast. Approximately 1.5 litres of the final, composted swine manure was filtered through wire mesh and then through cheesecloth to remove large particles. The filtrate was centrifuged at 8,000 RPM in 250 mL bottles for 15 minutes to remove bacterial cells and small particles. A GSA rotor and a Beckman R-5 centrifuge were used. The supernatant was extracted overnight with an equal volume of ethyl acetate. The ethyl acetate was decanted and evaporated to give an extract. The composted manure was extracted overnight with an equal volume of hexane. The hexane was decanted and evaporated to give an extract. The cells and small particles recovered from the centrifugation step were also extracted with ethyl acetate and hexane and the solvents evaporated off to give 2 extracts. Extracts were applied to paper discs and the solvent was allowed to evaporate. The discs were then applied to the surface of Petri plates inoculated with Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, and Corynebacterium xerosis. For antifungal activity discs were applied to plates of potato-dextrose agar swabbed with spores of Fusarium sp., Mucor plumbens and Penicillium crysogenum. Plates were incubated and zones of growth inhibition around each disc were recorded. As noted in the interim report extracts of commercial composts inhibited Gram-positive bacteria but not Gram-negative bacteria and yeast. After 18 weeks
of composting extracts of swine manure were inhibitory to the growth of a variety
of bacteria and fungi. Most of the inhibitory activity was in the liquid of the
composted swine manure not in the cells (presumably bacteria) and small
particles which were separated by centrifugation. The ethyl acetate extract of the
liquid compost was the most inhibitory extract. The ethyl acetate extract inhibited
the growth of all bacteria, both Gram-positive and Gram-negative and the three
fungi tested. The compost is still more inhibitory towards Gram-positive bacteria
than Gram-negative bacteria. The ethyl acetate extract of the liquid compost and
the hexane and ethyl acetate extracts of the cells in the comost were inhibitory
towards Staphylococcus aureus 43300, a methicillin-resistant (MRSA) strain. In
contrast to the extracts of commercial composts the ethyl acetate extract of
composted swine manure was inhibitory towards fungi. In particular the ethyl
acetate extract of composted swine manure was inhibitory towards a test
Fusarium species.
Table 1. Inhibition of growth of bacterial pathogens by compost extracts.


NT = not tested
- = no inhibition, + = inhibition of growth
Table 2. Inhibition of growth of fungi by compost extracts.
Early results from this project demonstrated that extracts of composts are inhibitory towards microorganisms. This may be related to the observation that some types of compost are inhibitory towards plant pathogens though this has been investigated only for fungal pathogens. Results from this study demonstrate that composted swine manure contains compounds which are inhibitory towards some types of fungi and bacteria. Therefore application of composted swine manure to agricultural soils may affect the microbial ecology of the soil. This may be beneficial since fungal pathogens such as Fusarium could be inhibited. Fungi are the most important plant pathogens. Wheat is an important crop in Manitoba and Fusarium head blight is common especially in the more moist parts of the province. Head blight is economically important as it destroys wheat or lowers the quality of the grain. Fusarium spores overwinter in the soil before infecting a new crop. Composted swine manure or the antifungal compound(s) in composted swine manure may represent a potential treatment for this important problem. The extracts of the liquid component of composted swine manure and, to a lesser extent, the extracts of the cells and small particles in the manure inhibited the growth of Gram-positive bacteria more strongly than Gram-negative bacteria. Given the difference in inhibition on 2 different groups of bacteria by the extracts it is possible that the application of composted swine manure to soils may change the bacterial ecology of the soil. Bacteria play many important roles in soil including nitrogen fixation, denitrification, mobilization of other nutrients, breakdown and recycling of carbon compounds, plant pathogenicity and protection from the pathogenicity of other microorganisms. Changing the ecology of the soil by the application of composted swine manure may have both desirable and undesirable consequences. The consequences of the application of composted swine manure on the microbial ecology of agricultural soils should be examined. The majority of antibacterial activity and all the antifungal activity were found in the extracts of the liquid composted manure. However some antibacterial activity was seen in the extracts of the material removed from the compost by centrifugation. This is the small particles and bacterial and fungal cells. It is well known that bacteria and fungi produce antibiotics. Presumably the antibacterial and antifungal activity in both types of extracts comes from these cells, that is the antimicrobial activity is produced by and secreted from bacteria or fungi present in the manure. Further work is needed to determine whether the antimicrobial activity is truly from the bacteria or fungi in the manure or from the manure itself. If the activity comes from bacteria or fungi these could be isolated and examined to determine whether a commercially viable process can be developed to produce antibiotics. An antifungal compound would be especially valuable given the scarcity of antifungal antibiotics. The majority of antibacterial activity and all the antifungal activity were found in the ethyl acetate extracts. Hexane is a much more non-polar solvent than ethyl acetate. This means the antimicrobial compound(s) responsible for growth inhibition of the bacteria and fungi is relatively polar. In addition no inhibitory activity was found in the methanol extracts meaning the antimicrobial compound(s) are not extremely polar. This moderate degree of polarity is a feature of most antibiotics. If the compound was too polar it would not cross over the cellular membrane to attack targets inside the cell and if the compound was too non-polar it would not dissolve into water in concentrations high enough to inhibit any microorganism. In conclusion there are compounds in composted swine manure which inhibit the growth of fungi and bacteria. These compounds may play a role in the microbial ecology of soil which receives an application of composted swine manure. A potentially commercial antifungal agent may be present in the composted manure.


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