Performing Department
(N/A)
Non Technical Summary
Cattle production is the most economically important US agricultural activity, but the industry is under pressure to improve sustainability as beef production uses more land and energy and has a higher global warming potential than other major protein sources. Improving feed efficiency is arguably the best approach for enhancing sustainability, as much of the cost and environmental footprint of cattle production is due to feed production. Though numerous factors influence cattle feed efficiency, many recent studies have demonstrated that rumen microbiome composition plays a key role. Bacteriophages (viruses that only infect bacteria) are promising tools for altering microbiomes, but their stringent host-specificity has hindered their widespread adoption. Yet contrary to the common belief that bacteriophages are highly specific, we recently developed methods to isolate wide host-range (polyvalent) bacteriophages, and have found them to be highly effective for microbial control. Advantages of polyvalent over narrow host-range bacteriophages include decreased phage cocktail complexity, higher titers in environments with multiple hosts (e.g., rumen), decreased phage decay rates, enhanced biofilm propagation, and more economical production.The goal of this project is to develop polyvalent bacteriophage libraries for rumen microorganisms associated with cattle feed efficiency, including Methanobrevibacter ruminantium, Anaerovibrio lipolyticus, Fusobacterium sp., and Streptococcus bovis. Bacteriophages will be isolated using specialized methods that select for those with a predetermined host-range based on rumen microbiome composition. Each bacteriophage will then be characterized and tested for efficacy within a bioreactor operated to simulate the cattle rumen. Bacteriophage cocktails will be formulated and tested for their ability to minimize the development of bacteriophage resistance. Successful completion of this project is anticipated to lead to a library of bacteriophages that can selectively control the growth of several bacteria that negatively impact feed efficiency. These bacteriophages may then be further developed as livestock feed additives that can be utilized to improve animal feed efficiency, which consequently reduces production costs and improves environmental sustainability.
Animal Health Component
20%
Research Effort Categories
Basic
20%
Applied
20%
Developmental
60%
Goals / Objectives
The overarching goal of the proposed research is to develop phage libraries that can control the abundance of select rumen microbial species that are associated with feed efficiency phenotypes in cattle. Individually or in combination, such libraries will serve as the basis for a series of minimum viable products (MVPs) needed to conduct proof-of-concept studies and obtain customer validation. The key objectives we seek to accomplish are:1. Identify rumen microbial genera and species associated with feed efficiency phenotypes that are most permissive to phage infection.Hypothesis: Bacteria and archaea will vary in their susceptibility to phage infection (and ease of phage isolation) which will greatly influence the time needed to isolate phages.Significance: The time needed to isolate phages for a bacterial species can vary from days to years. Thus, from a commercialization perspective, it is important to identify the more permissive hosts so that initial efforts can be concentrated on those species most likely to yield successful results in a short timeframe (8 months).2. Assess the commercial practicality of using predefined phage cocktails for rumen microbiome manipulation.Hypothesis: Polyvalent phages will facilitate the use of active treatment strategies against rumen bacteria or archaea without the need for prior knowledge of microbiome composition.Significance: Active treatment would allow much lower phage doses to be used, reducing production costs 1000-fold or more relative to passive treatment. Moreover, the number of distinct phages per cocktail is constrained by dose requirements and economic considerations. While it is only practical to use a few phages in a cocktail intended for passive treatment, hundreds of different phages could potentially be used in a cocktail intended for active treatment, allowing much broader host coverage.
Project Methods
The methods being utilized for this project include:Selective bacterial isolation.It is important to have a representative library of bacterial strains that the bacteriophages may encounter during implementationand that can be used for assessing bacteriophage host range. As normally only a few strains of any species are commercially-available, we will utilize selective isolation procedures (enrichment cultures, selective plate media) to obtain pure stains of each target bacterial and archaeal species.A major source for isolates will be rumen fluid samples, which will be obtained from a processing plant, or from cannulated cattle at academic or agricultural research facilities.M. ruminantiumwill be isolated from rumen samples by preparing enrichment cultures with SAB medium amended with streptomycin, penicillin, and vancomycin, and an 80% H2/20% CO2atmosphere.Fibrobacter succinogenesstrains will be isolated using cotton enrichment cultures. Anaerovibriostrains will be isolated using linseed oil-rumen fluid-agar.Fusobacteriumwill be isolated using PY broth containing 50 mM lactate or lysine, followed by plating on JVN agar.Enterococcosel broth will be used for the isolation of newS. bovisstrains. In each case, approximately 1 g rumen digesta will be suspended in 10 mL media and briefly shaken. Aliquots will then be added by syringe to pre-sterilized Hungate tubes containing culture medium with an appropriate anaerobic gas headspace, followed by incubation at 39 °C until visible growth occurs. Samples will then be spread on agar plates and incubated in an anaerobic container until colonies form. Colony PCR and full-length 16S rRNA gene sequencing will be used for species identification.Bacteriophage pool preparation. We have adapted commonly used methods for phage pool preparation to reduce bias and achieve maximum diversity.Passage through 0.22 µm filters and the addition of chloroform are often used to remove bacterial contaminants from phage pools, but select against large and lipid-containing phages, respectively. Both are known to be present in the rumen and are widespread in gut environments. An additional consideration for reducing pool bias is the buffer used for extracting phages. For example, a wide range of bacteriophage isolation methods rely on the use of 10 mM sodium pyrophosphate, but this same concentration was found to reduce the viability of phage suspensions by 7 x 10-4in just 30 mins, and is actually used in protocols to generate phage deletion mutants. Potassium citrate buffers are also commonly used for eluting phages, but citrate has been reported to rapidly inactivate numerous phages. Thus, we use 250 mM glycine or 10% beef extract solutions, which more effectively maintain phage viability. Phages will be detached from soil and other solid particles by shaking in 250 mM glycine buffer (pH 8). Low speed centrifugation is used to remove larger particles and bacteria. The sample is then split into several aliquots, and subjected to various pool preparation methods, including direct enrichment, direct precipitation with polyethylene glycol 8000 (PEG), 0.45 μm filtration, and 0.22 µm filtration. Phage filtrates are concentrated byPEGprecipitation and resuspended in SM buffer. The 0.45 µm filtrate is split and half treated with chloroform. In this manner, we are able to recover a greater fraction of phage diversity, and also have some pools that have been purified of all bacterial contaminants. We have also developed a novel method for directly isolating phages for a specific host directly from an environmental sample (e.g., rumen and fecal samples) that is difficult to filter due to the presence of high molecular weight substances. We synthesized superparamagnetic nanoparticles (SNPs) conjugated to vancomycin (to promote binding to bacterial cell walls) and PEG (to increase binding to Gram-negative bacteria). These modified SNPs are then bound to an excess of the target host by incubation, and directly added to the environmental sample. Host-specific phages in the sample bind to the bacteria-SNP conjugates and can be harvested and concentrated magnetically.Bacteriophage isolation and characterization.We utilize three primary strategies for phage isolation, including direct plating, enrichment followed by plating, and prophage induction. To minimize labor and resource use, each phage pool is initially tested for lytic activity by spotting serial dilutions onto an agar overlay of the target host. If clearance is observed, plaque assays are used to isolate individual phages, which are then purified by repetitively streaking on host lawns until a uniform plaque morphology is achieved. If no clearance is observed from any pools created from a sample, rationally-selected pools from that sample are subjected to enrichment, and the enriched pools are then tested for lysis after purification and concentration. Additionally, enrichment cultures are prepared using the environmental sample (rather than pure cultures) with conditions selected to enhance the growth of the target host. For example, to increase the abundance of nativeFusobacteriumspecies within a rumen sample, we introduce small amounts into a minimal media containing either 50 mM lysine or lactate. In general, we have found that successfully enriching in this mannerresults in the isolation of large numbers of phages relative to amendment with an exogenous strain, though in some cases the latter is necessary.Two modified sequential, multi-host isolation methods develop by us will also be adapted to anaerobic conditions, and used to isolate phages. Method Ais used for bacterial hosts that form good lawns in agar overlays, while method Bwas designed for use with hosts that do not. These methods are biased towards the isolation of polyvalent phages because they include steps for the dilution of rapidly growing narrow host-range phages, and require growth on multiple hosts.Phages will be purified by plate streaking, and host-ranges will be verified using double-layer plate assays with each host.Prophage induction and adaptation.Isolating prophages by induction from bacterial genomes is highly advantageous forfastidiousand anaerobic bacteria, where the isolation of strictly lytic phages has proven to be extremely difficult. Moreover, temperate phages are known to outnumber lytic phages in the rumen. To induce prophages, cultures of individual strains are either subjected to heat treatment or exposed to sublethal levels of mitomycin C or norfloxacin until a decrease in optical density is observed. Phages are then harvested and characterized as usual.While temperate phages are not typically desired for microbial control applications, it is possible to isolate lytic mutants of temperate phages that have naturally-occurring deletions in genes known to be involved in the maintenance of lysogeny or in bacterial virulence. By doing so, temperate phages become lytic and can be used as any other natural strictly lytic phage. This method vastly enhances our ability to construct diverse phage cocktails that can infect a wide range of host strains.Evaluation of efficacy.Phage cocktail efficacy on selected rumen targets and potential off-target effects will be assessed using anaerobic batch cultures inoculated with rumen fluid and amended with phage cocktails. To ensure the presence of target hosts, some batches will be amended with either 105or 106cells/mL. Cocktails will be diluted such that individual phages will be added to batch cultures at concentrations of 0, 105or 108phages/mL. After 2 or 6 h incubation (108and 105phages/mL respectively), samples from each batch will be DNase treated to remove all extracellular DNA, heat-inactivated, subjected to standard DNA extraction, and analyzed by both RT-qPCR (for total 16S rRNA) and 16S rRNA gene sequencing.