Extraction of Microbial DNA from Breast Milk

Microbial DNA was extracted from 231 breast milk samples using a modified protocol from the DNeasy Powerfood Microbial kit (Qiagen, UK) (formerly PowerFood™ Microbial DNA Isolation kit, MoBio, Carlsbad, CA) as previously outlined^{22 (link)}. Briefly, milk samples were subjected to initial centrifugation of 4000g × 30 min at 4 °C, the fat layer removed with a sterile cotton swab (Thermo Fisher Scientific, Inc.), and the supernatant was discarded. Cell pellets were washed twice with phosphate buffered saline (Sigma Aldrich) and treated with 90 µL of 50 mg/mL lysozyme (Sigma Aldrich) and 50 µL of 5 KU/mL mutanolysin (Sigma Aldrich) followed by incubation at 55 °C × 15 min. Samples were subsequently treated with 28 µL of 20 mg/mL proteinase k (Qiagen, UK) and incubated further at 55 °C for 15 min followed by using the DNeasy Powerfood Microbial kit protocol. Negative controls using sterile molecular water (Sigma Aldrich) were extracted as above.