Bovine milk microbiota: Evaluation of different DNA extraction protocols for challenging samples

Abstract

The use of an adequate protocol that accurately extracts microbial DNA from bovine milk samples is of importance for downstream analysis such as 16S ribosomal RNA gene sequencing. Although sequencing platforms such as Illumina are very common, there are reservations concerning reproducibility in challenging samples that combine low bacterial loads with high amounts of host DNA. The objective of this study was to evaluate six different DNA extraction protocols applied to four different prototype milk samples (low/high level of colony‐forming units [cfu] and somatic cells). DNA extracts were sequenced on Illumina MiSeq with primers for the hypervariable regions V1V2 and V3V4. Different protocols were evaluated by analyzing the yield and purity of DNA extracts and the number of clean reads after sequencing. Three protocols with the highest median number of clean reads were selected. To assess reproducibility, these extraction replicates were resequenced in triplicates (n = 120). The most reproducible results for α‐ and β‐diversity were obtained with the modified DNeasy Blood & Tissue kit after a chemical pretreatment plus resuspension of the cream fraction. The unmodified QIAamp DNA Mini kit performed particularly weak in the sample representing unspecific mastitis. These results suggest that pretreatment in combination with the modified DNeasy Blood & Tissue kit is useful in extracting microbial DNA from challenging milk samples. To increase reproducibility, we recommend that duplicates, if not triplicates, should be sequenced. We showed that high counts of somatic cells challenged DNA extraction, which shapes the need to apply modified extraction protocols.

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

“…Lastly, it should be noted that while these steps worked well for the analysis of bulk milk from holding and processing vats, other bovine milk sample types may require different methodological approaches for microbial DNA extraction and analysis. In particular, there are certain challenges when using milk collected directly from individual healthy or mastitic cow quarters or teat canals [ 20 , 63 ]. For those milk samples, it is also important to consider the difficulties for aseptic collection, avoiding teat canal, udder skin and environmental microorganisms [ 20 , 64 , 65 ].…”

“…In particular, there are certain challenges when using milk collected directly from individual healthy or mastitic cow quarters or teat canals [ 20 , 63 ]. For those milk samples, it is also important to consider the difficulties for aseptic collection, avoiding teat canal, udder skin and environmental microorganisms [ 20 , 64 , 65 ]. Compared to bulk milk, freshly expressed milk may have higher bovine cell numbers or can be enriched with bacteria which are no longer dominant after storage or processing.…”

“…Compared to bulk milk, freshly expressed milk may have higher bovine cell numbers or can be enriched with bacteria which are no longer dominant after storage or processing. Milk collected directly from the teat may also contain compounds that inhibit PCR and therefore result in the need for different DNA extraction and purification steps [ 19 , 20 ].…”

“…Challenges to the examination of the bovine milk microbiota are notable because even high-quality bulk milk, containing low numbers of total bacteria (10 3 to 10 4 cells/ml) still contains a diverse microbiota with many bacterial species [16][17][18]. The nuance to this issue is expanded even further when evaluating freshly expelled milk from individual healthy and mastitic cows which appear to have DNA recalcitrant to PCR amplification [19,20]. These characteristics and the presence of high levels of proteins and fats in milk show the need for optimized bacterial DNA extraction protocols.…”

A culture-, amplification-independent, and rapid method for identification of pathogens and antibiotic resistance profile in bovine mastitis milk

“…host cell/DNA exists. Most previous research that used PF (Ganda et al, 2016;Lima et al, 2018;Taponen et al, 2019;Al-Harbi et al, 2021;Ma T. et al, 2021;Schwenker et al, 2022) or NG kit (Quigley et al, 2012;Cheema et al, 2021;Lyons et al, 2021) considered 16S metagenomic sequencing for bacterial identification following DNA extraction; therefore, no host DNA contamination was reported. While amplicon sequencing is a sensitive method for taxonomy clarification, the relevance of this approach for rapid diagnostic and AMR gene detection in mastitis is questionable.…”