An acute dietary intervention in lactation has variable impacts on the maternal and infant microbiota and milk antimicrobial proteins

Abstract

The first years of life are a critical period for microbial colonisation of the infant gut, which has been linked with health and disease outcomes in later life. Breastfeeding has been shown to have a significant impact on both composition and function of the infant gut microbiome. This is due to vertical transmission of microbes and exposure to other non-microbial factors, such as antimicrobial proteins, in human milk (HM). Given the importance of HM and the infant gut microbiome for lifelong health, this thesis investigated factors that influence HM composition (both microbial and non-microbial), with a focus on maternal diet as a modifiable determinant. Further, by utilising an acute maternal dietary intervention during lactation, the significance of maternal diet for infant gut composition and function was explored. While many studies have assessed the determinants of the HM microbiota, this has not been investigated in an Australian population. To address this gap, we first collected HM samples from a small cohort (n=29) of Australian mothers to characterise HM bacterial profiles. We investigated potential associations between HM bacterial composition and maternal factors, such as body mass index (BMI), mode of delivery, breast pump use, allergy, and parity, and infant factors, such as sex, mode of feeding, pacifier use, and introduction of solids. Our findings confirmed previous reports that maternal BMI, mode of delivery, and breast pump use are associated with HM bacterial profiles. Additionally, we found that infant pacifier use was associated with an altered HM microbiome, the first report of such a finding. With a more complete picture of the HM microbiome in Australian women and its key determinants, we next sought to focus on maternal diet and its impact on the maternal and infant microbiota, as diet is the single biggest modifiable determinant of the human microbiome. Previous observational studies have linked maternal diet with the HM microbiota. Such studies have also associated maternal diet during pregnancy with the infant gut microbiota. However, the impact of diet during lactation on the maternal and infant microbiota has not been examined. To address this gap, we performed an interventional study, in which exclusively breastfeeding mothers (n=11) were placed on a low-sugar, low-fat, high-fibre diet for two weeks. We analysed maternal faecal and HM samples collected immediately before, immediately after, and 4-8 weeks after the intervention. Full-length 16S rRNA gene sequencing was used for high-resolution bacterial profiling. Additionally, infant faecal samples were collected for shotgun metagenomic sequencing immediately pre- and postintervention to examine the effect of maternal diet on both composition and function of the infant gut microbiome. The intervention had no significant impact on infant gut microbiome composition; however, it was associated with significant changes in the functional capacity of the infant gut microbiome. The intervention also elicited small changes in a low number of bacterial taxa in both the maternal faecal and HM microbiota, some of which persisted 4-8 weeks post-intervention. Given that the dietary intervention shifted the function, but not the composition, of the infant gut microbiome, we next investigated whether the diet modulated other biologically relevant components in HM, namely the antimicrobial proteins lysozyme and lactoferrin. However, given that little is known about daily, circadian, and between-breast variations in the concentrations of lysozyme and lactoferrin, the stability of these proteins in mature HM needed to first be established. HM samples were collected from 18 mothers who undertook our acute dietary intervention across a 3-week period; daily, three times a day, and from both breasts. Both lactoferrin and lysozyme showed small, but statistically significant, variability over time, with significant day-to-day and circadian differences; however, no significant variation was detected between breasts. These findings underscore the need to standardise sample collection time, or to collect samples at multiple time points throughout the day, in order to obtain reliable estimation of HM lysozyme/lactoferrin content. The concentration of HM lysozyme was reduced during the first week of the dietary intervention, while HM lactoferrin levels were lower in the second week of the intervention. Thus, maternal diet may impact HM antimicrobial protein concentrations. We hypothesise that this may be due to dietary effects on maternal systemic inflammation. Together, these data demonstrate the importance of maternal factors, including BMI and maternal diet and infant feeding practices, including breast pump and pacifier use, as potential modifiers of HM microbial composition. In addition, a controlled maternal dietary intervention elicited small but significant changes to the maternal faecal and HM microbiota, and to HM antimicrobial proteins. While these changes were associated with significant alterations to the functional potential of the infant gut microbiome, they did not affect its composition. The data presented in this thesis therefore highlight potential limitations of maternal diet as a target to improve infant health via manipulation of the microbiome.