Validated methods are urgently needed to improve DNA sequence-based assessments of complex bacterial communities. In this study, we used 16S rRNA PCR amplicon and gDNA mock community standards, consisting of nine, dairy-associated bacterial species, to evaluate the most commonly applied 16S rRNA marker gene DNA sequencing and analysis platforms used in evaluating dairy and other bacterial habitats. Our results show that bacterial metataxonomic assessments are largely dependent on the DNA sequencing platform and read curation method used. DADA2 improved sequence annotation compared with QIIME 1, and when combined with the Ion Torrent PGM DNA sequencing platform and the Greengenes database for taxonomic assignment, the most accurate representation of the dairy mock community standards was reached. This approach will be useful for validating sample collection and DNA extraction methods and ultimately investigating bacterial population dynamics in milk- and dairy-associated environments.

We set out to identify the viable and total bacterial content in milk as it passes through a large-scale, dairy product manufacturing plant for pasteurization, concentration, separation, blending, and storage prior to cheese manufacture. A total of 142 milk samples were collected from up to 10 pieces of equipment for a period spanning 21 h on two collection dates in the spring and late summer of 2014. Bacterial composition in the milk was determined by 16S rRNA marker gene, highthroughput DNA sequencing. Milk samples from the late summer were paired such that half were treated with propidium monoazide (PMA) to enrich for viable cells prior to quantification by PCR and identification by DNA sequence analysis. Streptococcus had the highest median relative abundance across all sampling sites within the facility on both sampling dates. The proportions of Anoxybacillus, Thermus, Lactococcus, Lactobacillus, Micrococcaceae, and Pseudomonas were also elevated in some samples. Viable cells detected by PMA treatment showed that Turicibacter was enriched after high-temperature short-time pasteurization, whereas proportions of Staphylococcus were significantly reduced. Using clean-in-place (CIP) times as a reference point, Bacillus, Pseudomonas, and Anoxybacillus were found in high relative proportions in several recently cleaned silos (Ͻ19 h since CIP). At later times (Ͼ19 h after CIP), 10 of 11 silos containing elevated viable cell numbers were enriched in Acinetobacter and/or Lactococcus. These results show the tremendous point-to-point and sample-dependent variations in bacterial composition in milk during processing. IMPORTANCE Milk undergoes sustained contact with the built environment during processing into finished dairy products. This contact has the potential to influence the introduction, viability, and growth of microorganisms within the milk. Currently, the population dynamics of bacteria in milk undergoing processing are not well understood. Therefore, we measured for total and viable bacterial composition and cell numbers in milk over time and at different processing points in a cheese manufacturing facility in California. Our results provide new perspectives on the dramatic variations in microbial populations in milk during processing even over short amounts of time. Although some of the changes in the milk microbiota were predictable (e.g., reduced viable cell numbers after pasteurization), other findings could not be easily foreseen based on knowledge of bacteria contained in raw milk or when the equipment was last cleaned. This information is important for predicting and controlling microbial spoilage contaminants in dairy products.

Antimicrobial resistance (AMR) represents a considerable burden to health care systems, with the public health community largely in consensus that AMR will be a major cause of death worldwide in the coming decades. Humans carry antibiotic resistance in the microbes that live in and on us, collectively known as the human microbiome.

Ophiocordyceps sinensis is a highly valued fungus that has been used as traditional Asian medicine. This fungus is one of the most important sources of income for the nomadic populations of the Tibetan Plateau. With global warming and excessive collection, the wild O. sinensis resources declined dramatically. The cultivation of O. sinensis hasn’t been fully operational due to the unclear genetic basis of the fruiting body development. Here, our study conducted pairwise comparisons between transcriptomes acquired from different growth stages of O. sinensis including asexual mycelium (CM), developing fruiting body (DF) and mature fruiting body (FB). All RNA-Seq reads were aligned to the genome of O. sinensis CO18 prior to comparative analyses. Cluster analysis showed that the expression profiles of FB and DF were highly similar compared to CM. Alternative splicing analysis (AS) revealed that the stage-specific splicing genes may have important functions in the development of fruiting body. Functional enrichment analyses showed that differentially expressed genes (DEGs) were enriched in protein synthesis and baseline metabolism during fruiting body development, indicating that more protein and energy might be required for fruiting body development. In addition, some fruiting body development-associated genes impacted by ecological factors were up-regulated in FB samples, such as the nucleoside diphosphate kinase gene (ndk), β subunit of the fatty acid synthase gene (cel-2) and the superoxide dismutase gene (sod). Moreover, the expression levels of several cytoskeletons genes were significantly altered during all these growth stages, suggesting that these genes play crucial roles in both vegetative growth and the fruiting body development. Quantitative PCR (qPCR) was used to validate the gene expression profile and the results supported the accuracy of the RNA-Seq and DEGs analysis. Our study offers a novel perspective to understand the underlying growth stage-specific molecular differences and the biology of O. sinensis fruiting body development.