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Untargeted sequencing of nucleic acids present in food can inform the detection of food safety and origin, as well as product tampering and mislabeling issues. The application of such technologies to food analysis may reveal valuable insights that are simply unobtainable by targeted testing, leading to the efforts of applying such technologies in the food industry. However, before these approaches can be applied, it is imperative to verify that the most appropriate methods are used at every step of the process: gathering of primary material, laboratory methods, data analysis, and interpretation. The focus of this study is on gathering the primary material, in this case, DNA. We used bovine milk as a model to (i) evaluate commercially available kits for their ability to extract nucleic acids from inoculated bovine milk, (ii) evaluate host DNA depletion methods for use with milk, and (iii) develop and evaluate a selective lysis-propidium monoazide (PMA)-based protocol for host DNA depletion in milk. Our results suggest that magnetically based nucleic acid extraction methods are best for nucleic acid isolation of bovine milk. Removal of host DNA remains a challenge for untargeted sequencing of milk, highlighting the finding that the individual matrix characteristics should always be considered in food testing. Some reported methods introduce bias against specific types of microbes, which may be particularly problematic in food safety, where the detection of Gram-negative pathogens and hygiene indicators is essential. Continuous efforts are needed to develop and validate new approaches for untargeted metagenomics in samples with large amounts of DNA from a single host. IMPORTANCE Tracking the bacterial communities present in our food has the potential to inform food safety and product origin. To do so, the entire genetic material present in a sample is extracted using chemical methods or commercially available kits and sequenced using next-generation platforms to provide a snapshot of the microbial composition. Because the genetic material of higher organisms present in food (e.g., cow in milk or beef, wheat in flour) is around 1,000 times larger than the bacterial content, challenges exist in gathering the information of interest. Additionally, specific bacterial characteristics can make them easier or harder to detect, adding another layer of complexity to this issue. In this study, we demonstrate the impact of using different methods for the ability to detect specific bacteria and highlight the need to ensure that the most appropriate methods are being used for each particular sample.

Culture and screening of gut bacteria enable testing of microbial function and therapeutic potential. However, the diversity of human gut microbial communities (microbiota) impedes comprehensive experimental studies of individual bacterial taxa. Here, we combine advances in droplet microfluidics and high-throughput DNA sequencing to develop a platform for separating and assaying growth of microbiota members in picoliter droplets (MicDrop). MicDrop enabled us to cultivate 2.8 times more bacterial taxa than typical batch culture methods. We then used MicDrop to test whether individuals possess similar abundances of carbohydrate-degrading gut bacteria, using an approach which had previously not been possible due to throughput limitations of traditional bacterial culture techniques. Single MicDrop experiments allowed us to characterize carbohydrate utilization among dozens of gut bacterial taxa from distinct human stool samples. Our aggregate data across nine healthy stool donors revealed that all of the individuals harbored gut bacterial species capable of degrading common dietary polysaccharides. However, the levels of richness and abundance of polysaccharide-degrading species relative to monosaccharide-consuming taxa differed by up to 2.6-fold and 24.7-fold, respectively. Additionally, our unique dataset suggested that gut bacterial taxa may be broadly categorized by whether they can grow on single or multiple polysaccharides, and we found that this lifestyle trait is correlated with how broadly bacterial taxa can be found across individuals. This demonstration shows that it is feasible to measure the function of hundreds of bacterial taxa across multiple fecal samples from different people, which should in turn enable future efforts to design microbiota-directed therapies and yield new insights into microbiota ecology and evolution. IMPORTANCE Bacterial culture and assay are components of basic microbiological research, drug development, and diagnostic screening. However, community diversity can make it challenging to comprehensively perform experiments involving individual microbiota members. Here, we present a new microfluidic culture platform that makes it feasible to measure the growth and function of microbiota constituents in a single set of experiments. As a proof of concept, we demonstrate how the platform can be used to measure how hundreds of gut bacterial taxa drawn from different people metabolize dietary carbohydrates. Going forward, we expect this microfluidic technique to be adaptable to a range of other microbial assay needs., (Copyright © 2020 Villa et al.)