Table to farm: Using shotgun metagenomics to quantify antibiotic resistance on farms feeding food scraps to laying hens

Table to farm: Using shotgun metagenomics to quantify antibiotic resistance on farms feeding food scraps to laying hens

Summary

The objective of this project is to provide poultry farmers and industry workers with preliminary insight into the effects of incorporating food scraps into an on-farm composting system. The primary goal is to perform shotgun metagenomic sequencing in order to identify and quantify antibiotic resistance genes (ARGs) present in various products and intermediaries on a poultry farm currently accepting food scraps, ranging from the scraps themselves to final consumer products such as eggs and home gardening supplies. This project emerged in response to new legislation, Act 148 in Vermont, which dictates the diversion of food scraps for agricultural practices such as poultry feed. However, the persistence of resistance genes throughout this cycle is currently unknown and may pose a risk in the burgeoning fight against global antibiotic resistance. Additionally, on integrative farms such as the one sampled here, the potential for introduction of novel resistance (human-associated) into the farm setting and amplification of resistance through the spread back to consumers the need for investigations such as this is paramount. Due to concerns with coverage from sequencing and utility of results, work will now be conducted on a single farm but expanded to ten sample types from this location to allow for a more comprehensive view of the dynamics of ARG transfer throughout the composting process. Sampling began in October 2015, and final samples will be acquired in January 2017 for sequencing later in the month. Samples will include raw food waste and manures, various stages of the composting process, and consumer products such as eggs and worm castings. As this particular substrate has not been sequenced in this way before, we have had to develop new methods for DNA extraction to limit the contamination of samples by plant and human genetic material, but is still on track for completion by July 2017.

Objectives/Performance Targets

Below are the originally stated objectives, as well as modifications that have been made to address the new sampling scheme:

1.     Develop a novel screening method to determine the prevalence of ARGs in unprocessed food waste received by selected poultry farms in Vermont.

Specific aims of this objective include:

a) Identify target genes of interest for screening and create a comprehensive reference database for sequencing,

b) Identify target genes with known primers for validation of metagenomic analysis,

c) Identify antimicrobial resistance genes (ARGs) in food waste samples taken from poultry farms, and

d) Categorize the types and abundance of ARGs identified in food waste samples obtained from poultry farm(s).

The aims and goals of this objective have largely remained the same, however due to limitations of sequencing and difficulty of DNA extractions the number and types of samples have been changed slightly. Concerns over plant material contaminating products for shotgun sequencing, and subsequently reducing the coverage for antibiotic resistance genes of interest, have led to adjustments in extraction techniques. In order to combat this and sequence the best product possible, two major changes have been made. Firstly, pre-processing steps for the samples have been added to limit Eukaryotic cells present in the product to be extracted. For this aim, each sample will be filtered through a 20-micron filter, which should selectively allow for bacterial cells and extracellular DNA only to pass through. Secondly, the use of multiple DNA extraction methods will be employed to identify which yields the highest quality results, confirmed by performing two rounds of quantitative real time-PCR (qRT-PCR): 16S which amplifies Prokaryotic DNA, and 18S which amplifies Eukaryotic DNA. This will allow us to estimate the relative bacterial: plant content in each sample before it is submitted for sequencing, and select the method that yields the lowest plant contamination. Ideally, this will significantly improve results overall and allow for the most comprehensive findings.

A further change proposed is the sampling scheme. Previously, 4 sample types were to be taken from 3 farms at two different time points, for a total of 24 samples. After consultation with the sequencing facility, it was suggested that the sampling scheme be revised to reduce the total number of samples, as the depth at this sample number would be unlikely to give us the resolution needed to properly quantify ARGs present. By reducing the sampling to 10 samples from a single farm, we can afford to purchase an additional lane for sequencing (5 samples/lane instead of 24 samples/lane) and significantly improve our results. Overall, this should not affect our ability to accomplish the aims under this objective, but will shift the scope to allow for a more complete view of the trends on a poultry farm already practicing food waste composting and feeding.

2.     Quantify the diversity of food waste management practices being implemented on integrated composting and poultry farms in Vermont. In the face of growing antimicrobial resistance concerns, characterizing farmers’ current awareness and efforts to prevent ARG introduction and spread is critical. For the 3 study farms, we will complete a survey to assess the level of knowledge about proper food waste handling and identify measures already being taken to prevent the spread or growth of antimicrobial resistance.

Specific aims include:

a) Assess the level of knowledge of poultry farmers about limiting antimicrobial resistance and

b) Identify measures utilized at the farm level to reduce antimicrobial resistance.

Again, these aims will be slightly changed to accommodate the new sampling strategy. Namely, instead of addressing a survey across farms, we will focus our questionnaire to be more relevant to the practices of the single farm and will have greater context for our results. In addition to concerns over sampling, our objectives now better suit the needs of the farmer as well. During initial conversations the potential for these results to impact the regulation of these waste management practices without full context could limit their future use if the risk is not fully investigated. The aim of this project is risk management, not banning the use of food scraps as feed and composting substrate. Food waste acceptance and management represents a significant portion of farmer income as both a service and value-added product. Through the comprehensive sampling we will now perform, we are in a better position to assess the fate of resistance genes in this system and stand to be able to create more specific recommendations for future work.

Briefly, with the revised sampling strategy we anticipate to complete the original objectives with slight modifications. In summary, we anticipate to be able to quantify the presence and any changes in antibiotic resistance genes throughout the composting cycle; from the acceptance of food scraps from various commercial facilities, critical stages in the composting process, substances fed directly to poultry, and to the finished product that would re-enter the consumer cycle. In addition to this data about the samples themselves, we will use surveys and conversations with stakeholders to assess their knowledge of antibiotic resistance and risk management, as well as their aims and the impacts of food scrap acceptance on their future production and success. These objectives will all be completed by this spring and should be ready to present in early summer through our outreach efforts.

Accomplishments/Milestones

Objective 1a has been completed. Reference databases from OneCodex and CosmosID platforms will be used to categorize ARGs present in the samples. Objectives 1c and 1d will be performed as soon as the best method of DNA extraction has been identified, and objective 1b will be completed once these results have been summarized and the 8-10 most frequently found genes will be chosen for confirmation of shotgun analysis.

At this point in the project, major accomplishments include establishing methodologies for sample processing. Due to the complexity of both extractions and sequencing, initial steps of the project have centered more on method development. This delay has set back the anticipated timeline slightly, but should not impact the date of completion. However, this leaves us with the potential more applicable data for outreach and risk management purposes and ultimately may be much more useful to farmers participating in food waste acceptance programs. Upon completion of the objectives stated above, we hope to assess the fate of ARGs in the consumer-agricultural pipeline and drive future experiments and recommendations for handling these products.

Impacts and Contributions/Outcomes

Currently there are no specific results to report. However, this study has the potential to describe the fate of antibiotic resistance genes in a farm setting at a depth that has never been accomplished before. While the scope is limited to a single farm at this point, it establishes the necessary protocols and methodologies that will streamline future work as well as provide baseline data for future comparisons. Additionally, this data may inform future handling and management practices for food scraps. For example, if food scraps from a specific industry or source carry a significantly higher “load” of resistance genes, these could be handled differently or composted at separate facilities to avoid transfer to agricultural settings. It may also identify areas for more targeted future studies, such as waste-stream specific sampling or expansion to other agricultural industries that may use these products. 

Collaborators: