Maternal HIV infection and the milk microbiome

Early infancy is a critical period for immune development. In addition to being the primary food source during early infancy, human milk also provides multiple bioactive components that shape the infant gut microbiome and immune system and provides a constant source of exposure to maternal microbiota. Given the potential interplay between allergic diseases and the human microbiome, this study aimed to characterise the milk microbiome of allergic mothers. Full-length 16S rRNA gene sequencing was performed on milk samples collected at 3 and 6 months postpartum from 196 women with allergic disease. Multivariate linear mixed models were constructed to identify the maternal, infant, and environmental determinants of the milk microbiome. Human milk microbiome composition and beta diversity varied over time (PERMANOVA R2 = 0.011, p = 0.011). The season of infant birth emerged as the strongest determinant of the microbiome community structure (PERMANOVA R2 = 0.014, p = 0.011) with impacts on five of the most abundant taxa. The milk microbiome also varied according to the type of maternal allergic disease (allergic rhinitis, asthma, atopic dermatitis, and food allergy). Additionally, infant formula exposure reduced the relative abundance of several typical oral taxa in milk. In conclusion, the milk microbiome of allergic mothers was strongly shaped by the season of infant birth, maternal allergic disease phenotype, and infant feeding mode. Maternal allergic disease history and infant season of birth should therefore be considered in future studies of infant and maternal microbiota. Trial Registration: ClinicalTrials.gov identifier: ACTRN12606000281594.

Chapter 12 - Maternal Factors Related to Variability in the Human Milk Microbiome

Introduction Human milk once considered sterile is a habitat for a diverse microbiome. Breast milk is vital in the formation of the newborn microbiome and affects various health outcomes. Its microbiota is influenced by mode of delivery, geographic location, mastitis, diet, obesity, and gestational age and lactation stage. Knowledge of region-wise data regarding human milk microbiome is the need of an hour. Purpose The aim of this study was to isolate and characterize breast milk microbiome and to know the effect of gestational age and lactation stage on it. Methods A prospective longitudinal study included 30 mothers, of whom 10 were of gestational age < 32 weeks, 10 between 32 and 36 6/7 weeks, and the remaining 10 above 37 weeks of gestation at the time of delivery. Milk samples were collected and analyzed for microbiome at three different time points, that is, colostrum, transition milk (7-9 days), and mature milk (after 14 days). Results 381bacterial colonies were isolated and 242 colonies were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and cross-verified with 16s RNA sequencing, which included 38 different bacterial species. The majority of colonies (229/242) identified belonged to 4 phyla- firmicutes 119 (51.97%), proteobacteria 48 (20.96%), Bacillota 41 (17.9%), and actinobacteria 21 (9.17%). Bacterial diversity increased with gestational age and lactation stage and the proportion of actinobacteria and Bacillota increased significantly with lactation stage ( P value −.039), showing that preterm mother’s milk has a lesser quantity of beneficial bacteria. Conclusion This study provided the insight into effect of gestational age and lactation stage on breast milk microbiome. The diversity of milk microbiome increases with lactation and gestational age; however, the preterm mother’s milk lacks sufficient beneficial bacteria.

Emerging frontiers in human milk microbiome research and suggested primers for 16S rRNA gene analysis

Background: The human milk microbiome has been somewhat characterized, but little is known about changes over time and relations with maternal factors such as nutrient intake.Objective: We sought to characterize the human milk microbiome and described associations with maternal nutrient intake, time postpartum, delivery mode, and body mass index (BMI; in kg/m2).Methods: Milk samples (n = 104) and 24-h diet recalls were collected 9 times from 21 healthy lactating women from day 2 to 6 mo postpartum. Women were classified by BMI as healthy weight (<25) or overweight or obese (≥25). Bacterial taxa were characterized with the use of the high-throughput sequencing of the 16S ribosomal RNA gene.Results: The milk microbiome was relatively constant over time, although there were small changes in some of the lesser-abundant genera. Relative abundances of several taxa were associated with BMI, delivery mode, and infant sex. For instance, overweight and obese mothers produced milk with a higher relative abundance of Granulicatella than did healthy-weight women (1.8% ± 0.6% compared with 0.4% ± 0.2%, respectively; P < 0.05). Relative abundances of several bacterial taxa were also associated with variations in maternal dietary intake. For example, intakes of saturated fatty acids (rs = −0.59; P = 0.005) and monounsaturated fatty acids (rs = −0.46; P = 0.036) were inversely associated with the relative abundance of Corynebacterium; total carbohydrates (rs = −0.54; P = 0.011), disaccharides (rs = −0.47; P = 0.031), and lactose (rs = −0.51; P = 0.018) were negatively associated with Firmicutes; and protein consumption was positively correlated with the relative abundance of Gemella (rs = 0.46; P = 0.037).Conclusions: Factors associated with variations in the human milk microbiome are complex and may include maternal nutrient intake, maternal BMI, delivery mode, and infant sex. Future studies designed to investigate the relation between maternal nutrient intake and the milk microbiome should strive to also evaluate dietary supplement usage and analyze the collected milk for its nutrient content.

Growth faltering is common in Guatemalan indigenous communities, but the possibility that it may be related to milk microbial composition has not been explored. For this cross-sectional study, unrelated mother–infant dyads (n = 64) from eight communities in the remote Western Highlands of Guatemala were recruited. Milk samples and infant length-for-age and weight-for-age Z-scores were collected at two stages of lactation: early (6–46 days postpartum, n = 29) or late (109–184 days postpartum, n = 35). Within each stage of lactation, infants were subdivided into mildly underweight [weight-for-age Z-score (WAZ) < –1 SD] or normal weight (WAZ ≥ –1 SD) and mildly stunted [length-for-age Z-score (LAZ) < –1.5 SD] or non-stunted (LAZ ≥ –1.5 SD). 16S ribosomal RNA gene sequencing was used to identify milk microbial communities, and DESeq2 was used to compare the differential abundance (DA) of human milk microbiota at the species level for WAZ and LAZ subgroups at each stage of lactation. A total of 503 ESVs annotated as 256 putative species across the 64 human milk samples were identified. Alpha diversity did not differ, but beta-diversity redundancy analysis identified four distinct clusters among the four WAZ (p = 0.004) and LAZ subgroups (p = 0.001). DA identified 15 different taxa in the WAZ and 11 in the LAZ groups in early lactation and 8 in the WAZ and 19 in the LAZ groups in late lactation. Mothers’ milk had more DA taxa of oropharyngeal and environmental bacteria with opportunistic activities in the LAZ < –1.5 SD infants, whereas the LAZ ≥ –1.5 SD had DA taxa with potential probiotic and antimicrobial inhibitory activity against pathogens. In particular, milk microbial communities of infants not classified as underweight or stunted had more beneficial species including Lactococcus_lactis. These findings suggest the potential associations between the milk microbiome at the species level with infant growth prior to 6 months of age. These data provide important evidence of the associations between the human milk microbiome and the growth of breastfed infants.

We analysed the human milk microbiome in a cohort of 80 lactating women and followed the dynamics in taxa over the course of lactation from birth to 6 months. Two hundred and thirty one milk samples were collected from full-term lactating women at 1, 4, 8 and 24 weeks following birth and analysed for microbiota composition using 16S rRNA sequencing. A significant decrease in milk microbiota diversity was observed throughout the first 6 months of lactation, with the greatest difference seen between week 8 and week 24. Nine genera predominated in milk over lactation from week 1 to week 24, comprising of Staphylococcus, Streptococcus, Pseudomonas, Acinetobacter, Bifidobacterium, Mesorhizobium, Brevundimonas, Flavobacterium, and Rhodococcus; however, fluctuations in these core genera were apparent over time. There was a significant effect of stage of lactation on the microbiome, while no effect of birth mode, infant sex and maternal BMI was observed throughout lactation. Streptococcus had the highest mean relative abundance at week 1 and 24 (17.3% and 24% respectively), whereas Pseudomonas predominated at week 4 (22%) and week 8 (19%). Bifidobacterium and Lactobacillus had the highest mean relative abundance at week 4 (5% and 1.4% respectively), and occurred at a relative abundance of ≤ 1% at all other time points. A decrease in milk microbiota diversity throughout lactation was also observed. This study concluded that lactation stage was the primary driving factor in milk microbiota compositional changes over lactation from birth to 6 months, while mode of delivery was not a factor driving compositional changes throughout human lactation.

Our goal was to determine trends in low birth weight and preterm birth among US infants born to HIV-infected women. We used data from the longitudinal Pediatric Spectrum of HIV Disease, a large HIV cohort, to assess trends in low birth weight and preterm birth from 1989 to 2004 among 11,321 study infants. Among women with prenatal care, we also assessed risk factors, including maternal antiretroviral therapy during pregnancy, that were predictive of low birth weight and preterm birth using univariate and multivariate logistic regression models. Overall, 11,231 of 14,464 infants who were enrolled in Pediatric Spectrum of HIV Disease were tested during the neonatal period. From 1989 to 2004, testing increased from 32% to 97%. The proportion of HIV-exposed infants who had low birth weight decreased from 35% to 21% and occurred in all racial/ethnic groups. Prevalence of preterm birth decreased from 35% to 22% and occurred in all groups. Any maternal antiretroviral therapy use increased from 2% to 84%. Among 8793 women who had prenatal care, low birth weight was associated with a history of illicit maternal drug use, unknown maternal HIV status before delivery, symptomatic maternal HIV disease, black race, Hispanic ethnicity, and infant HIV infection. Antiretroviral therapy or lack of it was not associated with low birth weight. Among women with prenatal care, preterm birth was associated with a history of illicit maternal drug use, symptomatic maternal HIV disease, no antiretroviral therapy, receipt of a 3-drug highly active antiretroviral therapy regimen with protease inhibitors, black race, and infant HIV infection. The proportion of infants who had low birth weight or were born preterm declined during an era of increased maternal antiretroviral therapies. These Pediatric Spectrum of HIV Disease trends differ from the overall increases in both outcomes among the US population.

Evidence on the beneficial effects of microorganisms in human milk is emerging. Obesity and gestational diabetes mellitus appear to be related to alterations in the maternal gut microbiota and human milk composition. However, knowledge about the influence of maternal obesity and gestational diabetes mellitus on the human milk microbiota remains limited. To summarize and systematically assess the evidence on the influence of obesity and gestational diabetes mellitus on the diversity and/or composition of the human milk microbiota. A systematic review following PRISMA guidelines was conducted. Searches were performed in the MEDLINE, Cochrane Database of Systematic Reviews, Web of Science, and EMBASE, as well as in ClinicalTrials.gov and the World Health Organization International Clinical Trials Registry Platform. The ROBINS-E tool was used to assess the risk of bias. Out of 1,473 studies identified and 24 protocol records, eight studies were selected for final analysis. Exposure to gestational diabetes mellitus and maternal obesity were associated with changes in the human milk microbiota's diversity and/or composition. Lactating women exposed to obesity had a higher prevalence of Staphylococcus and a lower prevalence of Bifidobacterium in their milk. Those exposed to gestational diabetes mellitus had a higher prevalence of Staphylococcus, Gemella, and Prevotella. Findings regarding bacterial phyla prevalence were inconclusive. Based on the findings presented in this systematic review, it is not yet possible to draw definitive conclusions regarding the true influence of maternal obesity and/or gestational diabetes mellitus on the diversity or composition of the human milk microbiota.

Chapter 17 - Human Milk Microbes – Summary and Research Gaps

Infant growth trajectory may influence later-life obesity. Human milk provides a wide range of nutritional and bioactive components that are vital for infant growth. Compared to formula-fed infants, breastfed infants are less likely to develop later-onset obesity, highlighting the potential role of bioactive components present in human milk. Components of particular interest are the human milk microbiota, human milk oligosaccharides (HMOs), short-chain fatty acids (SCFAs), and antimicrobial proteins, each of which influence the infant gut microbiome, which in turn has been associated with infant body composition. SCFAs and antimicrobial proteins from human milk may also systemically influence infant metabolism. Although inconsistent, multiple studies have reported associations between HMOs and infant growth, while studies on other bioactive components in relation to infant growth are sparse. Moreover, these microbiome-related components may interact with each other within the mammary gland. Here, we review the evidence around the impact of human milk microbes, HMOs, SCFAs, and antimicrobial proteins on infant growth. Breastfeeding is a unique window of opportunity to promote optimal infant growth, with aberrant growth trajectories potentially creating short- and long-term public health burdens. Therefore, it is important to understand how bioactive components of human milk influence infant growth.

Healthcare workers are now being targeted for marketing of diagnostic tools for mastitis that were developed for the dairy industry and which aim to provide information regarding choice of antibiotic treatment. Meanwhile, scientists are striving to understand how the human microbiome affects health and wellbeing and the importance of maintenance of bacterial balance in the human body. Breast milk supplies a multitude of bacteria to populate the baby’s intestinal tract and kick-start the immune system. Researchers propose a paradigm shift in the understanding of bacterial content in breast milk and an alternative paradigm for the understanding of lactational mastitis: there is the beginning of evidence that many cases of lactational mastitis will resolve spontaneously. An international group of researchers is attempting to answer how dietary habits, birth mode, genetics and environmental factors may impact the bacterial content of breast milk. Until we have more comprehensive knowledge about the human milk microbiome, diagnostic aids for identification of women in need of antibiotic therapy for mastitis remain unreliable. Diagnostic aids could lead to the injudicious use of antibiotic therapy, which in turn may rob the infant of bacteria valuable for development of its immune system. The marketing of diagnostic aids for use in human medicine, that were originally developed for use in cows, is neither evidence-based nor good ethical practice.

Human milk contains a dynamic and complex site-specific microbiome, which is not assembled in an aleatory way, formed by organized microbial consortia and networks. Presence of some genera, such as Staphylococcus, Streptococcus, Corynebacterium, Cutibacterium (formerly known as Propionibacterium), Lactobacillus, Lactococcus and Bifidobacterium, has been detected by both culture-dependent and culture-independent approaches. DNA from some gut-associated strict anaerobes has also been repeatedly found and some studies have revealed the presence of cells and/or nucleic acids from viruses, archaea, fungi and protozoa in human milk. Colostrum and milk microbes are transmitted to the infant and, therefore, they are among the first colonizers of the human gut. Still, the significance of human milk microbes in infant gut colonization remains an open question. Clinical studies trying to elucidate the question are confounded by the profound impact of non-microbial human milk components to intestinal microecology. Modifications in the microbiota of human milk may have biological consequences for infant colonization, metabolism, immune and neuroendocrine development, and for mammary health. However, the factors driving differences in the composition of the human milk microbiome remain poorly known. In addition to colostrum and milk, breast tissue in lactating and non-lactating women may also contain a microbiota, with implications in the pathogenesis of breast cancer and in some of the adverse outcomes associated with breast implants. This and other open issues, such as the origin of the human milk microbiome, and the current limitations and future prospects are addressed in this review.

Ross, Amanda; Raab, Gillian M.; Mok, Jacqueline; Gilkison, Sara; Hamilton, Barbara; Johnstone, Frank D. Author Information

The standard treatment for high-grade squamous intraepithelial lesions is excisional involving the uterine cervix, while surveillance is an acceptable approach for low-grade squamous intraepithelial lesions. There is controversy about excisional treatment on pregnancy outcomes. The objective of this study was to determine pregnancy outcomes in women living with and without HIV who underwent excisional treatment for high-grade cervical intraepithelial lesions. This retrospective cohort study compared the pregnancy outcomes of women with and without HIV who were or were not treated for cervical intraepithelial lesions. A cohort of 488 women with and without HIV infection who did or did not receive excisional treatment for cervical intraepithelial lesions between 2009 and 2022 was enrolled. Adverse pregnancy outcomes (preterm delivery and pregnancy loss) in women with and without HIV, untreated or treated for cervical dysplasia, were recorded and analysed. The significance of the obtained results was judged at the 5% level. The study was conducted at all Academic Model Providing Access to Healthcare-Kenya satellite sites, which offer cervical cancer screening and treatment for cervical dysplasia in western Kenya. The Moi Teaching and Referral Hospital was also included. A cohort of 488 women aged between 20 years and 49 years, with and without HIV, diagnosed and treated for high-grade cervical intraepithelial neoplasia, and those followed up for low-grade cervical intraepithelial neoplasia between 2009 and 2022, were included. The study was interested in adverse pregnancy outcomes, particularly pregnancy loss and preterm delivery following cervical excision treatment for high-grade cervical intraepithelial lesions. After adjustment for confounding factors, excisional treatment involving the uterine cervix-particularly cold knife conisation-was associated with higher odds of adverse pregnancy outcomes (OR 13.1; 95% CI 1.1 to 137.1; p=0.032). A prior history of adverse pregnancy outcomes was also strongly associated with subsequent adverse outcomes after treatment (OR 37.7; 95% CI 13.8 to 102.7; p<0.001). In contrast, maternal HIV infection was not independently associated with adverse pregnancy outcomes after adjustment (p=0.125). Adverse pregnancy outcomes after excisional treatment of the uterine cervix for high-grade squamous intraepithelial lesions are multifactorial and were associated with cold knife conisation and prior adverse pregnancy outcomes, while maternal HIV infection was not independently associated with adverse outcomes.