Fig 1.
Schematic diagram of the experimental design.
The BCMC was tested for the effects of variations in sample preparation, cell lysis, DNA purification and collection methods. Cells were lysed using a bead beater (B), vortex (V), or incubated for chemical lysis (C). Wash solution denotes the solutions included in DNA extraction kits used for purification. Wash step(s) were performed following manufacturer’s protocol. Underlined text indicates experiments performed with both BCMC1 and BCMC2. For all other experiments, BCMC2 was used. Filled boxes are methods used to test raw bulk milk samples.
Table 1.
Bacterial strains and expected relative abundances in the BCMC.
Table 2.
Cell lysis and DNA extraction methods tested.
Fig 2.
Comparison of BCMC cell enumeration methods.
The expected values are based on CFU enumeration for BCMC1 and direct counts by microscopy for BCMC2. The observed results represent the average of 15 replicates of BCMC1 and 33 replicates of BCMC2 according to 16S rRNA gene amplicon DNA sequencing. BCMC replicates were processed the same by MagMAX Total kit and lysed with bead beating at 6.5 m/sec for 1 min twice with 1 min interval on ice prior to PCR. Significant changes (DESeq2 adjusted p < 0.1 and log2 fold change > 1.5) compared to the expected values are indicated by the presence of asterisks.
Fig 3.
Microbial diversity and composition of BCMC taxa detected upon recovery from different volumes of milk.
BCMC2 was inoculated into UHT milk and sampled at the following volumes (cell numbers): 200 μL (1.26 × 103 cells), 1 mL (6.32 × 103 cells), 10 mL (6.32 × 104 cells), and 30 mL (1.90 × 105 cells). Expected percentages are estimated based on estimated cell numbers in the BCMC and 16S rRNA gene copy numbers for each species. The (A) total number of ASVs, (B) Bray-Curtis dissimilarity of intra-sample variation, and (C) expected taxa (9 bacterial species) are labeled with the corresponding taxonomic level from DNA sequencing results. Unexpected taxa were labeled as “Other”. Each bar represents a single replicate of BCMC2. Significant differences (Kruskal-Wallis with Dunn test, p < 0.05) from the expected value (A) and between sample groups (B) are indicated by the presence of asterisks.
Fig 4.
Relative proportions of taxa and UPGMA hierarchical clustering of BCMC exposed to different storage conditions.
UPGMA hierarchical clustering was based on Bray-Curtis dissimilarity matrix for (A) BCMC2 frozen with PBS or 25% v/v glycerol and (B) BCMC1 that were prepared fresh or exposed to five freeze-thaw cycles. Expected taxa (9 bacterial species) are labeled with the corresponding taxonomic level from 16S rRNA gene amplicon DNA sequencing results. Each bar represents a single replicate.
Fig 5.
Distances between the expected values and mock community samples.
(A) Bray-Curtis dissimilarity and (B) weighted UniFrac distances compared to expected values. Significant differences (Kruskal-Wallis with Dunn test, p < 0.05) from the standard DNA extraction method (Total kit with B1 lysis method) are indicated by the presence of asterisks. The following lysis methods were applied: B1 (bead-beating twice for 1 min at 6.5 m/sec with 1 min interval on ice), V1 (vortex at 1800 rpm for 15 min), C1 (incubation at 80°C for 20 min and 37°C for 60 min), B5 (bead-beating for 10 sec at 4 m/sec; C2 (incubation at 37°C for 60 min), and V3 (vortex at 1800 rpm for 30 sec). Total (MagMAX Total nucleic acid kit), Core (MagMAX Core DNA kit), Ultra2 (MagMAX Ultra 2.0 DNA kit); and PK (proteinase K). Abbreviations for the cell lysis and DNA purifications methods are also provided in Table 2. The letter R on the x-axis denotes RNase A treated samples.
Fig 6.
Effects of cell lysis methods on BCMC proportions.
The weighted difference was calculated using the following formula: (observed%—expected%) / expected%. Each circle is the average value from three replicates of BCMC2. Different colors indicate the value of weighted differences and dot sizes indicate the absolute values of weighted differences. Significant changes (DESeq2 adjusted p < 0.1 and log2 fold change > 1.5) compared to the expected values are indicated by the presence of asterisks. Abbreviations are used as follows (see also Table 2): B1 (bead-beating twice for 1 min at 6.5 m/sec with 1 min interval on ice), V1 (vortex at 1800 rpm for 15 min), C1 incubation at 80°C for 20 min and 37°C for 60 min); B5 bead-beating for 10 sec at 4 m/sec), C2 stands for incubation at 37°C for 60 min), and V3 (vortex at 1800 rpm for 30 sec). The letter “R” after lysing methods denotes RNase A treatment.
Fig 7.
Effects of B1, B2, B3, B4, V1, and V2 cell lysis methods on BCMC1 taxonomy.
DNA extractions were performed on BCMC1 using the MagMAX Total nucleic acid kit. Each bar represents the average value of three replicates. Significant differences (DESeq2 adjusted p < 0.1 and log2 fold change > 1.5) from the expected value are indicated by the presence of asterisks. Exp stands for expected, B1 (bead beat twice for 1 min at 6.5 m/sec with 1 min interval on ice), B2 (bead beat for 1 min at 6.5 m/sec), B3 (bead beat twice for 1 min at 3.5 m/sec with 1 min interval on ice), B4 (bead beat at 3.5 m/sec for 60 sec), V1 (vortex at 1800 rpm for 15 min), V2 (vortex at 1800 rpm for 10 min), and C1 (incubation at 80°C for 20 min and 37°C for 60 min).
Fig 8.
Beta diversity and weighted differences of commercial milk samples and the BCMC.
(A) weighted differences of mock community samples compared to the Total/B1 method, (B) Bray-Curtis dissimilarity, and (C) weighted UniFrac distances between milk samples are shown. Weighted differences for each organism are calculated using the following formula: (B5% or V3%—B1%) / B1%. Significant differences (B and C, Kruskal-Wallis with Dunn test, p < 0.05) are indicated by the presence of asterisks. Abbreviations are used as follows (see also Table 2): B1 (bead-beating twice for 1 min at 6.5 m/sec with 1 min interval on ice using the MagMAX Total kit), B5 (bead-beating for 10 sec at 4 m/sec), and (vortex at 1800 rpm for 30 sec). B5 and V3 samples were extracted using the MagMAX Total kit with PK digestion.