Abstract
ABSTRACTThe bovine udder is colonized by a huge quantity of microorganisms that constitute the intramammary ecosystem, with a specific role in modulating not only udder homeostasis and mastitis susceptibility, but also the quality of the dairy products. However, generating high-quality bacterial DNA can be critical, especially starting from a complex biological matrix like milk, characterized by high fat, protein, and calcium contents. Here, bacterial DNA was recovered from a commercial ultra-high-temperature (UHT) milk sample artificially spiked with a predetermined mock community composition and from three bulk tank milk (raw milk) samples. The DNA was isolated using three different protocols to evaluate the effect of the extraction procedures on the milk microbiota composition. In the mock community experiment, the bacterial profiles generated by the three DNA extraction protocols were profoundly different, with the genera Staphylococcus, Lactobacillus, Listeria, and Salmonella underestimated by all the protocols. Only one protocol revealed values close to the expected abundances for Escherichia/Shigella spp., Bacillus spp., Enterococcus spp., and Pseudomonas spp. On the other hand, the nonspiked UHT milk sample exhibited a similar microbiota composition, revealing the prevalence of Acinetobacter spp., for all the DNA extraction protocols. For the raw milk samples, the three DNA extraction kits performed differently, revealing significant separations in both the microbial richness (alpha diversity) and composition (beta diversity). Our study highlights the presence of significant differences among these procedures, probably due to the different DNA extracting capacities and to the different properties of the milk samples, revealing that the selection of DNA extraction protocol is a critical point.IMPORTANCE The advance of high-throughput technologies has increased our knowledge of the world of microorganisms, especially of microbial populations inhabiting living animals. This study provides evidence that milk, as other complex sources, could be critical for generating high-quality DNA for microbiota analysis. In addition, it demonstrates that the microbial population highlighted by metagenomic studies changes in relation to different DNA extraction procedures, revealing that attention should be paid especially when comparing different studies.
Highlights
The advance of high-throughput technologies has increased our knowledge of the world of microorganisms, especially of microbial populations inhabiting living animals
The average DNA yield and purity were analyzed before performing the analysis of the milk microbiota, for both the UHT and raw milk samples
Operational taxonomic unit (OTU) rarefaction curves based on the Chao1 metric reached a plateau after about 20,000 reads, suggesting that this depth of coverage was enough to describe the biological diversity within the samples
Summary
The advance of high-throughput technologies has increased our knowledge of the world of microorganisms, especially of microbial populations inhabiting living animals. The potential sources can be due to complex microbial environments, such as the bedding material, the milking equipment, the milker’s hand, or cross suckling [4] These microorganisms play a specific role in modulating the udder homeostasis and mastitis susceptibility and together with those acquired during the milking procedure, in influencing the quality of the dairy products [4, 5]. Generating high-quality bacterial DNA could be critical, especially starting from a matrix with high fat, protein, and calcium constituents [6]. These factors act as PCR inhibitors and can compromise the amplification of DNA. The performance and reproducibility of these protocols were assessed based on the quality and quantity of the DNA extracted and the bacterial community composition by performing 16S rRNAbased high-throughput sequencing on an ultra-high-temperature-treated (UHT) milk sample spiked with a defined mock community and three raw milk samples
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