Impact of expression mode and timing of sample collection, relative to milk ejection, on human milk bacterial DNA profiles.

AIM: To investigate the impact of expression mode: electric breast pump or hand expression, and timing of sample collection: pre- and post-milk ejection on human milk (HM) bacterial DNA profiles. METHODS AND RESULTS: Three HM samples from the same breast were collected from 30 breastfeeding mothers: a pre-milk ejection pump-expressed sample (pre-pump), a post-milk ejection pump-expressed sample (post-pump) and a post-milk ejection hand-expressed sample (post-hand). Full-length 16S rRNA gene sequencing was used to assess milk bacterial DNA profiles. Bacterial profiles did not differ significantly based on mode of expression nor timing of sample collection. No significant differences were detected in the relative abundance of any OTUs based on expression condition (pre-pump/ post-pump and post-pump/post-hand) with univariate linear mixed-effects regression analyses (all P-values > 0·01; α = 0·01). Similarly, no difference in richness was observed between sample types (number of observed OTUs: post-pump/post-hand P = 0·13; pre-pump/post-pump P = 0. 45). CONCLUSION: Bacterial DNA profiles of HM did not differ according to either expression method or timing of sample collection. SIGNIFICANCE AND IMPACT OF THE STUDY: Hand or pump expression can be utilized to collect samples for microbiome studies. This has implications for the design of future HM microbiome studies.

DNA extraction method influences human milk bacterial profiles.

AIMS: To evaluate four DNA extraction methods to elucidate the most effective method for bacterial DNA recovery from human milk (HM). METHODS AND RESULTS: Human milk DNA was extracted using the following methods: (i) Qiagen MagAttract Microbial DNA Isolation Kit (kit QM), (ii) Norgen Milk Bacterial DNA Isolation Kit (kit NM), (iii) Qiagen MagAttract Microbiome DNA/RNA Isolation Kit (kit MM) and (iv) TRIzol LS Reagent (method LS). The full-length 16S rRNA gene was sequenced. Kits MM and method LS were unable to extract detectable levels of DNA in 9/11 samples. Detectable levels of DNA were recovered from all samples using kits NM (mean = 0·68 ng µl-1 ) and QM (mean = 0·55 ng µl-1 ). For kits NM and QM, the greatest number of reads were associated with Staphylococcus epidermidis, Streptococcus vestibularis, Propionibacterium acnes, Veillonella dispar and Rothia mucilaginosa. Contamination profiles varied substantially between kits, with one bacterial species detected in negative extraction controls generated with kit QM and six with kit NM. CONCLUSIONS: Kit QM is the most suitable of the kits tested for the extraction of bacterial DNA from human milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Choice of extraction method impacts the efficiency of bacterial DNA extraction from human milk and the resultant bacterial community profiles generated from these samples.

Effects of maternal dietary egg intake during early lactation on human milk ovalbumin concentration: a randomized controlled trial.

BACKGROUND: There is limited understanding of how maternal diet affects breastmilk food allergen concentrations, and whether exposure to allergens through this route influences the development of infant oral tolerance or sensitization. OBJECTIVE: To investigate how maternal dietary egg ingestion during early lactation influences egg protein (ovalbumin) levels detected in human breastmilk. METHODS: In a randomized controlled trial, women were allocated to a dietary group for the first six weeks of lactation: high-egg diet (> 4 eggs per week), low-egg diet (one-three eggs per week) or an egg-free diet. Breastmilk samples were collected at 2, 4 and 6 weeks of lactation for the measurement of ovalbumin. The permeability of the mammary epithelium was assessed by measuring the breastmilk sodium : potassium ratio. Egg-specific IgE and IgG4 were measured in infant plasma at 6 weeks, and prior to the introduction of egg in solids at 16 weeks. RESULTS: Average maternal egg ingestion was associated with breastmilk ovalbumin concentration. Specifically, for each additional egg ingested per week, there was an average 25% increase in ovalbumin concentration (95% CI: 5-48%, P = 0.01). Breastmilk ovalbumin concentrations were significantly higher in the 'high-egg' group (> 4 eggs per week) compared with the 'egg-free' group (P = 0.04). However, one-third of women had no breastmilk ovalbumin detected. No detectable associations were found between mammary epithelium permeability and breastmilk ovalbumin concentrations. Infant plasma egg-specific IgG4 levels were also positively associated with maternal egg ingestion, with an average 22% (95% CI: 3-45%) increase in infant egg-specific IgG4 levels per additional egg consumed per week (P = 0.02). CONCLUSIONS AND CLINICAL RELEVANCE: Increased maternal egg ingestion is associated with increased breastmilk ovalbumin, and markers of immune tolerance in infants. These results highlight the potential for maternal diet to benefit infant oral tolerance development during lactation.

Bioimpedance spectroscopy in the infant: effect of milk intake and extracellular fluid reservoirs on resistance measurements in term breastfed infants.

BACKGROUND/OBJECTIVES: Bioimpedance spectroscopy is an accurate non-invasive method for measuring body composition in adults, but in infants it requires further testing and validation. Of the few studies of bioimpedance conducted in infants, none have comprehensively investigated the effect of milk intake volume. This study assessed the effect of the milk intake, feed duration and the volume of the infant's stomach and bladder on the resistance values pre-/post-feed to establish the feasibility of using these values interchangeably during data collection. SUBJECTS/METHODS: Forty-eight breastfeeding infants were measured at 2, 5, 9 and/or 12 months (n=62 sessions) within 1-2 min before the start and after the end of breastfeed. Median (IQR) time between measurements was 24 (20.0-30.0) min. Resistance measurements at 0 and 50 kHz, and infinite frequency (R0, R50 and Rinf) and resistance of intracellular water (Ricw) were analysed with customised infant settings. Milk intake was measured by test weights. Free-water volumes and free-water change were determined from stomach and bladder volumes calculated from ultrasound images. RESULTS: Small pre-to-post-feed changes (median (IQR): R0 -3.7 (-14.8, 14.3); R50 0.3 (-10.4, 15.0); Rinf 2.8 (-13.3, 35.5); Ricw 20.8 (-98.1, 290.9)) were not significantly different from zero (R0: P=0.92; R50: P=0.48; Rinf: P=0.32; Ricw: P=0.097). No significant effect of milk intake or free-water change was detected. CONCLUSIONS: The lack of consistent change in resistance across a breastfeed provides flexibility in the timing of measurements of infants in the research setting, such that typically pre- and post-feed measures of resistance can be used interchangeably.

Novel feeding system to promote establishment of breastfeeds after preterm birth: a randomized controlled trial.

OBJECTIVE: We aimed to determine if a novel feeding system where milk only flowed when the preterm infant created a vacuum would influence time to full oral feeds, the length of stay (LOS) in hospital and breastfeeding at discharge. STUDY DESIGN: This was a randomized controlled trial in the tertiary neonatal intensive care unit at King Edward Memorial Hospital, Perth, Australia. Eligibility criteria were: preterm infants of gestational age 25 to 34 weeks receiving >75% human milk by gastric tube. Infants were randomly assigned to being fed with a novel teat (NT) or conventional teat (CT). Intention to treat analysis was performed. RESULT: Time to full suck feeds was not different between groups. LOS was shorter (mean: 2.5 days; P=0.026) and less formula was fed at discharge in the NT group (P=0.036). CONCLUSION: Use of a NT that releases milk when the infant applies vacuum while establishing breastfeeding reduces duration of hospitalization of preterm infants.