Improved assessments of bulk milk microbiota composition via sample preparation and DNA extraction methods

Abstract

Although bacterial detection by 16S rRNA gene amplicon DNA sequencing is a widely-applied technique, standardized methods for sample preparation and DNA extraction are needed to ensure accuracy, reproducibility, and scalability for automation. To develop these methods for bovine bulk milk, we assembled and tested a bacterial cell mock community (BCMC) containing bacterial species commonly found in milk. The following protocol variations were examined:: BCMC enumeration (colony enumeration or microscopy), sample volume (200 μl to 30 ml), sample storage condition (frozen in PBS or 25% glycerol or exposure to freeze-thaw cycles), cell lysis method (bead-beating, vortex, enzymatic), and DNA extraction procedure (MagMAX Total, MagMAX CORE, and MagMAX Ultra 2.0, with and without either Proteinase K or RNase A). Cell enumeration by microscopy was more accurate for quantification of the BCMC contents. We found that least 10 mL (≥ 10 4 cells in high quality milk) is needed for reproducible bacterial detection by 16S rRNA gene amplicon DNA sequencing, whereas variations in storage conditions caused minor differences in the BCMC. For DNA extraction and purification, a mild lysis step (bead-beating for 10 s at 4 m/s or vortexing at 1800 rpm for 10 s) paired with the MagMAX Total kit and Proteinase K digestion provided the most accurate representation of the BCMC. Cell lysis procedures conferred the greatest changes to milk microbiota composition and these effects were confirmed to provide similar results for commercial milk samples. Overall, our systematic approach with the BCMC is broadly applicable to other milk, food, and environmental samples therefore recommended for improving accuracy of culture-independent, DNA sequence-based analyses of microbial composition in different habitats.

Improvement and Validation of a Genomic DNA Extraction Method for Human Breastmilk

“…DNA yield achieved a significant reduction (p =< 0.05) in Gram(+) (33.74 ± 25.83 ng/µL) compared with Gram(−) (80.68 ± 32.89 ng/µL) bacteria and yeasts (85.86 ± 34.84 ng/µL), suggesting that the difference in cell wall composition may have affected the DNA yields. In a previous study that evaluated the efficacy of different DNA extraction methods to reconstruct a microbiological profile, an underrepresentation of Gram(+) bacteria was observed [28,51], particularly bacteria of the genus Lactobacillus and Streptococcus, which have cell walls that are difficult to break down [31]. The results reported in a previous section suggest a significant decrease in the DNA extraction of Gram(+) bacteria (Figure 2a), which could explain the Gram(+) underrepresentation reported.…”

Host DNA depletion methods and genome-centric metagenomics of bovine hindmilk microbiome

“…Recently, several commercial kits have become available for depleting the host’s DNA from different niches including nasopharyngeal aspirate, saliva, human milk, and bovine milk ( 18 – 21 ). Regarding milk samples, the scientific literature reports the assessment of protocols considering bulk tanks or mock communities ( 19 , 22 ). However, these samples commonly have a higher microbial load than those found in hindmilk samples.…”