Dynamics of the infant gut microbiota in the first 18 months of life: the impact of maternal HIV infection and breastfeeding

Silvia Grant-Beurmann; Jibreel Jumare; Nicaise Ndembi; Olayemi Matthew; Ashley Shutt; Augustine Omoigberale; Olivia A. Martin; Claire M. Fraser; Man Charurat

doi:10.1186/s40168-022-01230-1

Dynamics of the infant gut microbiota in the first 18 months of life: the impact of maternal HIV infection and breastfeeding

Grant-Beurmann, Silvia; Jumare, Jibreel; Ndembi, Nicaise; Matthew, Olayemi; Shutt, Ashley; Omoigberale, Augustine; Martin, Olivia A.; Fraser, Claire M.; Charurat, Man 2022-04-12 00:00:00 Background: Access to antiretroviral therapy (ART ) during pregnancy and breastfeeding for mothers with HIV has resulted in fewer children acquiring HIV peri‑ and postnatally, resulting in an increase in the number of children who are exposed to the virus but are not infected (HEU). HEU infants have an increased likelihood of childhood infections and adverse growth outcomes, as well as increased mortality compared to their HIV‑unexposed (HUU) peers. We explored potential differences in the gut microbiota in a cohort of 272 Nigerian infants born to HIV ‑positive and nega‑ tive mothers in this study during the first 18 months of life. Results: The taxonomic composition of the maternal vaginal and gut microbiota showed no significant differ ‑ ences based on HIV status, and the composition of the infant gut microbiota at birth was similar between HUU and HEU. Longitudinal taxonomic composition of the infant gut microbiota and weight‑for ‑age z‑scores ( WAZ) differed depending on access to breast milk. HEU infants displayed overall lower WAZ than HUU infants at all time points. We observed a significantly lower relative abundance of Bifidobacterium in HEU infants at 6 months postpartum. Breast milk composition also differed by time point and HIV infection status. The antiretroviral therapy drugs, lamivudine and nevirapine, as well as kynurenine, were significantly more abundant in the breast milk of mothers with HIV. Levels of tiglyl carnitine (C5) were significantly lower in the breast milk of mothers without HIV. ART drugs in the breast milk of mothers with HIV were associated with a lower relative abundance of Bifidobacterium longum. Conclusions: Maternal HIV infection was associated with adverse growth outcomes of HEU infants in this study, and these differences persist from birth through at least 18 months, which is a critical window for the development of the immune and central nervous systems. We observed that the relative abundance of Bifidobacterium spp. was signifi‑ cantly lower in the gut microbiota of all HEU infants over the first 6 months postpartum, even if HEU infants were receiving breast milk. Breastfeeding was of benefit in our HEU infant cohort in the first weeks postpartum; however, ART drug metabolites in breast milk were associated with a lower abundance of Bifidobacterium. Keywords: HIV‑ exposed infants, Gut microbiota, Breast milk metabolome, Antiretroviral therapy, Breastfeeding, Bifidobacterium, Adverse growth outcome, Weight ‑for ‑age z‑score, Acylcarnitine, Kynurenine Background Improved access to antiretroviral therapy (ART) for *Correspondence: cmfraser@som.umaryland.edu Claire M. Fraser and Man Charurat contributed equally to this work as mothers with HIV during pregnancy and breastfeeding co‑senior authors. has resulted in fewer children acquiring HIV peri- and Department of Medicine, University of Maryland School of Medicine, postnatally [1]. There has been a resultant increase in the Baltimore, MD, USA Full list of author information is available at the end of the article number of children born to mothers with HIV who are © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 2 of 18 exposed to the virus, but who are not infected (HEU). weight was significantly lower among HEU babies (P < This population is estimated to be 14.8 million children 0.001), but the proportion of those born with low birth globally; with approximately 13.2 million children living weight (< 2.5 Kg) and those born prematurely did not in sub-Saharan Africa [2]. It has been shown previously differ significantly between HEU and HUU children. that HEU infants have an increased likelihood of child- Weight-for-age z-scores (WAZ) were significantly lower hood infections and adverse growth outcomes, as well as among HEU as compared to HUU babies at birth. The increased mortality compared to their HIV unexposed median duration of breastfeeding was 9 months, and (HUU) peers [3–7]. 46% of infants were exclusively breastfed for 6 months; Exposure to HIV in utero has been shown to impact however, in HEU infants there was a significantly the gut microbiota of HEU infants [8–11], pointing to a shorter duration of breastfeeding (P < 0.001) as well as potential link between maternal HIV status, the infant lower proportion of exclusively breastfed children (P < gut microbiota, and infant health. Perturbations in the 0.001). As expected, and consistent with standard rec- infant gut microbiota have been linked with altered ommendations, the use of TMP-SMX was significantly immunity, and increased susceptibility to disease [12– greater among HEU infants at all follow-up timepoints 16]. In addition, the breast milk microbiota has been (P < 0.001). This pattern was similar for overall antibi - previously found to differ between mothers with and otic prescription until the 9-month visit, beyond which without HIV [10] and survival of HEU infants in Africa the difference between HEU and HUU was not statisti - has been associated with breast milk oligosaccharide cally significant. The variable “antibiotic use” captured composition [17]. These breast milk oligosaccharides, in any antibiotic prescribed to the participants during their turn, have been linked to the gut microbiota of HEU [8]. clinic visit. It reflected trimethoprim-sulfamethoxazole Taken together, these findings provide considerable evi - (TMP-SMX), other antibiotics, or a combination of these, dence that maternal HIV status has a profound impact on thereby enabling us to account for any antibiotic use in the acquisition and subsequent development of the infant the analyses. During the 18-month study period, none of gut microbiota [18–21]. the infants included in this analysis became HIV-positive. We set out to further investigate the relationship between in utero HIV exposure and adverse growth out- Maternal vaginal and infant gut microbiomes increased comes in HEU by conducting a longitudinal study of the in diversity over time maternal and infant microbiota of 272 Nigerian mother- High-throughput sequencing of the hypervariable infant pairs, as well as the breast milk metabolome. We regions V3 and V4 of the 16S ribosomal RNA gene was hypothesized that acquisition of an altered gut micro- used to characterize the taxonomic composition of the biota from a mother with HIV, further exacerbated by samples collected in this study. Two sample types from differences in breast milk composition between mothers the mothers (vaginal swabs; MVS and stool; MST) were with and without HIV, negatively impacts growth and collected prior to (at time of enrollment, after 12 weeks increases the risk of adverse clinical outcomes among of gestation) and at birth (number of MST samples col- HEU infants. lected at birth was lower in comparison to prenatally due to fewer specimens being produced at that time point). Results Infant meconium (IMC) was collected at birth, and Most characteristics of mother‑infant pairs were similar infant stool samples (IST) were collected at 6 weeks, 6 regardless of motherʼs HIV status months, 9 months, 15 months, and 18 months postpar- Table 1 shows the baseline characteristics of the HEU tum (Table S1). and HUU infants and their mothers. The median age of We first looked broadly at the average microbiome mothers was 32 years, similar for women with and with- composition at each sample site over all time points. out HIV (P = 0.12). Most of the mothers were employed Principal coordinates analysis (PCoA) based on Bray- (87.1%), with no significant difference between the two Curtis dissimilarity revealed a distinct clustering of the groups (P = 0.853). The mothers of the HEU children mothers’ vaginal and stool samples (PERMANOVA, P were more likely to have lower levels of education (P < = 0.001; Fig. S1A). Consistent with this observation was 0.001), less likely to be married (P < 0.001), but more the finding that the alpha diversity between these sam - likely to be multiparous (P = 0.039). While there was a ples was significantly different with MST exhibiting a slightly greater proportion of deliveries via cesarean sec- higher diversity (Shannon index; SI = 4.05 ± 0.05 SEM) tion (33.8% vs. 28.5%) among the women with HIV, this in comparison to MVS (SI = 1.85± 0.04 SEM) (Fig. S2A; did not reach statistical significance (P = 0.418). The Shannon index, P < 0.001). Vaginal microbiome diver- women with HIV had a median CD4 count of 429 cells/ sity significantly increased after birth (Fig. 1A; Shan- ml (IQR: 285–566) at enrollment. The median birth non index, P < 0.001), while maternal stool microbiome Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 3 of 18 Table 1 Baseline maternal and infant characteristics N All HIV‑/HUU HIV+/HEU 131 141 P value Maternal Maternal Age (years), median (IQR) 263 32 (29, 36) 32 (29, 35) 32 (29, 37) 0.1248 Employed,n(%) 263 229 (87.1) 110 (86.6) 119 (87.5) 0.8527 Education,n(%) 263 < .0001 Primary/junior secondary 47 (17.9) 3 (2.4) 44 (32.4) Senior secondary 81 (30.8) 26 (20.5) 55 (40.4) Tertiary 135 (51.3) 98 (77.2) 37 (27.2) Married,n(%) 263 213 (81.0) 124 (97.6) 89 (65.4) < .0001 Parity,n(%) 262 0.0388 Primip 74 (28.2) 44 (34.9) 30 (22.1) 2–4 181 (69.1) 80 (63.5) 101 (74.3) > 4 7 (2.7) 2 (1.6) 5 (3.7) CD4 Count (cells/mm ), median (IQR) 136 428.5 (285, 566) Baby Gender, female,n(%) 272 118 (43.5) 60 (45.8) 58 (41.4) 0.4496 Delivery type,n(%) 272 0.4183 Vaginal 186 (68.4) 92 (70.2) 94 (66.7) Cesarean section 86 (31.6) 39 (29.8) 47 (33.3) Premature delivery,n(%) 268 20 (7.5) 7 (5.4) 13 (9.4) 0.2486 Birth weight (Kg), median (IQR) 271 3 (2.7, 3.4) 3.2 (2.8, 3.5) 2.9 (2.5, 3.25) < .0001 Birth weight <2.5 Kg,n(%) 271 33 (12.2) 12 (9.2) 21 (15.0) 0.1793 Anthropometrics at birth,z‑score, mean (SD) Weight‑for ‑age 271 − 0.71 (1.2) 0.39 (1.2) − 1.01 (1.2) < .0001 Meconium microbiome Shannon diversity, median (IQR) 176 1.75 (1.2, 2.3) 1.71 (1.2, 2.2) 1.78 (1.1, 2.3) 0.7386 Exclusive breastfeeding (6 months),n(%) 246 112 (45.5) 72 (62.1) 40 (30.8) < .0001 Breastfeeding duration (months), median (IQR) 272 6 (1, 9) 9 (9, 15) 1 (1, 6) < .0001 Trimethoprim‑sulfamethoxazole use (6 months),n(%) 246 131 (53.3) 15 (12.9) 116 (89.2) < .0001 Antibiotic use (6 months),n(%) 257 111 (43.2) 35 (29.7) 76 (54.7) < .0001 W F T Wilcoxon’s, Fisher’s, t test HUU, HIV‑unexposed uninfected; HEU, HIV ‑ exposed uninfected; IQR, interquartile range; N, number of participants; SD, standard deviation diversity was unchanged over this same time period between mothers with and without HIV for either of the (Fig. 1A; Shannon index, P = 0.138). sample sites (PERMANOVA, P = 0.876; Fig. S3). There The two sample types for the infants also displayed sig - were also no significant differences in bacterial diver - nificant differences (Fig. S1B). In the aggregate, IMC had sity identified based on HIV infection (Fig. S4; Shannon a lower diversity, (SI = 1.92 ± 0.05 SEM) compared to index, P = 0.672). IST (SI = 2.89 ± 0.02 SEM) (Fig. S2B; Shannon index, P < Longitudinal changes in the microbiota of mothers 0.001). As previously reported, we observed a significant with and without HIV were also investigated. No sig- increase in the diversity of IST from birth to 18 months nificant differences in the stool or vaginal microbiota postpartum (Fig. 1B; Shannon index, P < 0.001). between mothers with and without HIV (Fig. S5A) were found. The maternal stool microbiota contained both Maternal microbiome composition showed no significant Bacteroides and Prevotella from the phylum Bacteroi- differences based on HIV status detes, several genera in the Firmicutes phylum includ- To determine the impact of HIV infection on the mater- ing Blautia, Faecalibacterium, Lactobacillus, Roseburia, nal microbiota, the taxonomic composition of the micro- Staphylococcus and Streptococcus, and Bifidobacterium. biota from mothers with HIV was compared to that from The maternal vaginal microbiota was dominated by Lac - mothers without HIV. PcoA revealed no clear separation tobacillus, along with Gardnerella and Pseudomonas; Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 4 of 18 Fig. 1 Alpha diversity (Shannon index) for each sample type and timepoint. A Maternal stool (MST ) microbiome diversity did not change prenatally to birth (Shannon index, P = 0.138), whereas maternal vaginal (MSV ) microbiome diversity significantly increased after birth (Shannon index, P < 0.001). B Infant meconium (IMC) and infant stool (IST ) microbiome diversity displayed significant increases from birth to 18 months postpartum (Shannon index, P < 0.001). Horizontal lines in boxplots indicate median; boxes show first and third quartiles however, in mothers with HIV, the abundance of Lac- breast milk at these timepoints. As a result, the cohort tobacillus was lower (prenatal; 63% vs. 72%, birth; 51% was stratified not just by HIV exposure status, but also by vs. 55%) and the abundance of Gardnerella was higher breastfeeding status. (prenatal; 17% vs. 11%, birth; 13% vs. 12%) compared to In newborn infants that had not yet commenced mothers without HIV (Fig. S5B), however, these differ - breastfeeding, meconium samples were domi- ences did not reach statistical significance. At none of the nated by Pseudomonas, Enterobacter, Klebsiella, and timepoints was the bacterial diversity significantly differ - Corynebacterium. By contrast, the taxonomic compo- ent (Shannon index, P > 0.05) between mothers with and sition of all infant stool samples collected at various without HIV for any of the sample sites (Shannon index, time points postpartum was characterized by a high MVS—prenatal; P = 0.076, MVS—birth; P = 0.461, relative abundance of Bifidobacterium, Streptococcus, MST—prenatal; P = 0.761, MST – birth; P = 0.312, Fig. and Enterobacter. The relative abundances of several S5C). bacterial taxa significantly differed based on breast- feeding status as determined by MaAsLin2; Bifido- Breastfeeding status is associated with differences bacterium (FDR; P < 0.001) and Collinsella (FDR; P in the gut microbiota of infants = 0.040) were positively associated with breastfeed- At the time of this study, the World Health Organization ing [23], whereas Faecalibacterium (FDR; P = 0.007) (WHO) recommendation for mothers with HIV was to and Streptococcus (FDR; P = 0.803) were negatively exclusively breastfeed for 6 months; introduce comple- associated with breastfeeding. PcoA of stool samples mentary feeds afterwards, while continuing to breast- obtained from HEU vs. HUU infants exhibited no clear feed for up to 24 months [22]. This recommendation separation in any of the three breastfeeding groups was most relevant in situations where ART was avail- (PERMANOVA, P = 0.459; Fig. 2A); however, the able to guarantee the best chance for HIV-free survival Shannon diversity between breastfeeding HEU (SI = for exposed infants in resource-limited settings. How- 2.33 ± 0.05 SEM) and breastfeeding HUU (SI = 2.67 ± ever, many of the mothers with HIV in this study opted 0.04 SEM) was significantly different (Fig. 2B; Shannon not to breastfeed for long durations due to concerns for index, P < 0.001). There were no significant differences transmission of HIV to their babies (Table 1, Fig. S6). At between HEU (SI = 1.97 ± 0.07 SEM) and HUU (SI = 6 and 9 months postpartum, the majority (99% and 95%, 1.88 ± 0.07 SEM) infants’ alpha diversity at birth (not respectively) of HUU infants were still being breastfed, yet breastfeeding); similarly, there was no difference whereas only 39% and 17% of HEU infants were receiving between HEU (SI = 3.19 ± 0.04 SEM) and HUU (SI = Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 5 of 18 Breastfeeding HEU infants exhibit significantly higher 3.32 ± 0.06 SEM) alpha diversity in infants that are not weight‑for‑age z‑scores compared to non‑breastfeeding breastfeeding. None of the differences in relative abun- HEU infants at 6 weeks postpartum dance of bacterial taxa reached significance between Previous studies have shown that HEU infants have lower the microbiota of HEU and HUU infants within any of WAZ compared to HUU infants [27–30]. Our results the groups based on the breastfeeding status (Fig. 2C). are also consistent with those findings (Fig. 4A), which prompted us to further investigate whether there is a potential association between breastfeeding status and Breastfeeding infants from mothers with HIV exhibit WAZ in HUU and HEU infants. We ran linear regres- significantly less Bifidobacteria at 6 months postpartum sion models, followed by a pairwise post hoc test, which Previous research has shown the importance of breast- revealed that at 6 weeks postpartum, non-breastfeeding feeding and its ability to shape the gut microbiota in HEU exhibit significantly lower WAZ (− 1.82 ± 0.20 early life, both directly by exposure of the neonate to SEM) in comparison to breastfed HEU infants (− 0.99 ± the milk microbiota and indirectly, via maternal milk 0.11 SEM) (Fig. 4B, FDR; P < 0.001). That observation was factors that affect bacterial growth and metabolism no longer significant at 6 months postpartum or any later [24–26]. Because our data demonstrated that breast time points. The comparison of breastfeeding and non- feeding is associated with significant differences in breastfeeding HUU infants did not reveal any significant the diversity and composition of the gut microbiota differences at any time point. Comparing HUU with HEU in both HEU and HUU infants [8, 10], we performed by breastfeeding status and time point demonstrated that a cross-sectional comparison of the infant gut micro- HEU infants present with overall lower WAZ than HUU biota by time point. PCoA did not reveal any signifi- infants; newborn, breastfeeding, and non-breastfeeding cantly distinct clusters based on breastfeeding status HEU infants exhibit significantly lower WAZ at all time or time postpartum, although the distribution of data points (Fig. 4A, FDR; all P < 0.001). points was more dispersed in the breastfeeding cohort The gastrointestinal tract of full-term healthy infants (PERMANOVA, all P > 0.05; Fig. S7). This heteroge- is typically dominated by the genus Bifidobacterium neity in the distribution of data points correlated with [31–33]. Then, in the first months postpartum, the loss differences in the relative abundance of Bifidobacte- of Bifidobacterium species and/or gain of other bacte - rium longum (Fig. S8). Gut microbiota samples with ria can significantly alter the maturation of the micro - low B. longum clustered to the left of the PCoA, and bial community, which may lead to a variety of negative gut microbiota samples with high B. longum content consequences for host health including a predisposition were on the right. There were no significant differ- to autoimmune and metabolic diseases [34, 35]. Our ences in the Shannon diversity between HEU and HUU data revealed a significantly lower relative abundance of infant gut microbiota at any of the time points when Bifidobacterium in HEU infants at 6 months postpar - grouped by breastfeeding status (Fig. S9). We did, tum. Since breastfeeding is known to promote Bifido - however observe that one bacterial taxon, Bifidobacte- bacterium growth in the infant gut [33, 36], we explored rium, was significantly more abundant in the breast- whether the lack of breastfeeding in HEU infants during feeding HUU infants when compared to breastfeeding the first weeks and months postpartum was associated HEU infants at 6 months postpartum (Fig. 3A; FDR; with a lower abundance of Bifidobacteria. Indeed, we did P = 0.015). This difference was no longer observed in observe significantly lower relative abundances of Bifido - samples collected at 9, 15, and 18 months postpartum, bacteria in non-breastfed HEU infants at 6 weeks (15.80% which may, in part, reflect the introduction of solid ± 3.45% SEM) and 6 months (18.1% ± 1.90% SEM) post- foods at around 6 months of age. None of the taxa partum when compared to breastfeeding HEU infants at within the non-breastfeeding cohort reached statistical six weeks (34.93% ± 1.53% SEM) and 6 months (28.12% significance between HEU and HUU infants (Fig. 3B). (See figure on next page.) Fig. 2 The gut microbiota in breastfeeding infants from mothers with HIV differs from that seen in mothers without HIV. For these analyses, data across all time points were aggregated based on maternal HIV and/or breastfeeding status. A PCoA comparing HEU to HUU infants exhibited no significant separation within breastfeeding groups (PERMANOVA, P = 0.459). B The bacterial diversity of breastfeeding HEU infants (SI = 2.33 ± 0.05 SEM) and breastfeeding HUU infants (SI = 2.67 ± 0.04 SEM) showed a significant difference (Shannon index, P < 0.001). There were no significant differences in the Shannon diversity between HEU (SI = 1.97 ± 0.07 SEM) and HUU (SI = 1.88 ± 0.07 SEM) infants at birth (newborn); similarly, there was no differences between HEU infants (SI = 3.19 ± 0.04 SEM) and HUU infants (SI = 3.32 ± 0.06 SEM) infants that are not breastfeeding. C None of the bacterial taxa relative abundances differences reached significance between the microbiota of HEU and HUU infants (only genera made up with ASVs with a mean greater than 0.5% are shown) Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 6 of 18 Fig. 2 (See legend on previous page.) Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 7 of 18 Fig. 3 Breastfeeding infants from mothers with HIV exhibit significantly less Bifidobacteria at 6 months postpartum. A Bacterial taxa relative abundance differences between HEU and HUU infants within the breastfeeding cohort: Bifidobacterium was the only taxon that significantly differed between HEU and HUU infants at 6 months postpartum (6m pp, FDR; P = 0.015). B Bacterial taxa relative abundance differences between HEU and HUU infants within the non‑breastfeeding cohort: None of the bacterial taxa relative abundance differences in the non‑breastfeeding cohort reached significance between the microbiota of HEU and HUU infants. Because the majority of infants born to mothers without HIV were breastfed for 9 months, it was not possible to compare across non‑breastfeeding cohorts at these earlier time points. Only genera representing ASVs with a mean greater than 0.5% are shown at 6 weeks and 6 months postpartum. Table 2 shows that ± 2.29% SEM) (P < 0.001 and P = 0.008, respectively; the 34 mothers were a good representation of the entire Fig. 4C). cohort, with the exception of parity that was not signifi - To determine whether a low relative abundance of Bifi - cantly different between the two groups of mothers in dobacteria in HEU infants is a predictor of low WAZ, this subset, whereas it was significantly different in the we ran a logistic regression model with breastfeed- total cohort. ing included as a covariate. The model confirmed a link To analyze potential variations between the breast milk between low Bifidobacterium abundance and low WAZ of mothers with and without HIV, supervised analysis in HEU infants (β = 0.09, P = 0.018). with orthogonal partial least square discriminant analy- sis (OPLS-DA) and unsupervised analysis with princi- Breast milk composition differed by time point and HIV pal component analysis (PCA), were performed. A clear infection status separation between breast milk collected at 6 weeks and Breast milk metabolites from a subset of exclusively breast milk collected at 6 months was seen (not separated breastfeeding mothers with and without HIV were by HIV infection; Fig. S10A), which was also observed examined in more detail. Untargeted metabolite profil - in the PCA analysis (Fig. S10B). The metabolomics data ing was carried out using ultra-high-performance liquid were also analyzed by time point to identify potential chromatography/mass spectrometry/mass spectrom- differences by HIV infection. OPLS-DA showed a sepa - etry (UHPLC/MS/MS) to characterize a wide range of ration between the breast milk of mothers with and with- metabolites (a total of 553 compounds of known iden- out HIV (Fig. S11AB), however, that separation was not tity) present in breast milk samples from 34 mothers as strong in the PCA analysis and did not reach signifi - (Table 2; 17 exclusively breastfeeding mothers with HIV cance for either of the two time points (Fig. S11CD). and 17 exclusively breastfeeding mothers without HIV) Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 8 of 18 Fig. 4 Breastfeeding HEU infants exhibit significantly higher weight ‑for ‑age z‑scores compared to non‑breastfed HEU infants at 6 weeks postpartum. A HEU infants have significantly lower weight ‑for ‑age z‑scores ( WAZ) compared to HUU infants at all time points (FDR; P < 0.001). B At 6 weeks postpartum, non‑breastfeeding HEU infants exhibit significantly lower WAZ (− 1.82 ± 0.20) in comparison to breastfed HEU infants (− 0.99 ± 0.11) (FDR; P < 0.001). C Not breastfeeding HEU infants exhibited significantly lower relative abundances of Bifidobacteria at 6 weeks (15.80% ± 3.45% SEM) and 6 months (18.1% ± 1.90% SEM) postpartum when compared to breastfeeding HEU infants at 6 weeks (34.93% ± 1.53% SEM) and 6 months (28.12% ± 2.29% SEM) (P < 0.001 and P = 0.008, respectively) Two-way repeated measures analysis of variance Multivariate empirical Bayes analysis (MEBA) was used (ANOVA) (within subject) was used to identify signifi - to compare the time-course profiles between the breast cant differences in metabolites present in the breast milk milk of mothers with and without HIV. Metabolites with of mothers with and without at 6 weeks and 6 months high Hotelling’s T values comprise those whose profiles postpartum. ANOVA identified 106 metabolites that are more different between the breast milk of moth - significantly differed between the two groups and time ers with and without HIV across the time points. The points (FDR P < 0.05; Table S2). Among the 106 metab- 20 metabolites with the highest Hotelling’s T value are olites, 16 were associated with HIV infection, 88 were represented in Fig. 5 as a heatmap. The time course pro - associated with time point (6 weeks vs. 6 months post- files of the metabolites with the highest Hotelling’s T partum), and two were associated with both HIV infec- value are shown in Fig. S12. To determine whether the tion and time point. Within the 18 metabolites that were levels of any of these 20 metabolites are associated with significantly different between the breast milk of moth - the infants’ WAZ, Pearson’s correlations were run. After ers with and without HIV, 16 were higher and two were multiple comparison adjustment (FDR P value adjust- lower in the breast milk of mothers with HIV in com- ment set at 0.05), none of the 20 metabolites exhibited a parison to breast milk from mothers without HIV (Table significant correlation with WAZ. S2). The antiretroviral therapy (ART) drugs lamivudine and nevirapine were significantly more abundant in the Antiretroviral drugs found in breast milk of mothers breast milk of mothers with HIV. Kynurenine, which with HIV are associated with a lower relative abundance has received increasing attention due to its connection of Bifidobacterium longum to inflammation, the immune system, and neurological Previous studies have shown that antiretrovirals admin- conditions [37] was also significantly more abundant in istered to nursing mothers are present in their breast breast milk from mothers with HIV. Tiglyl carnitine (C5), milk; however, the degree of antiretroviral transfer from an acylcarnitine suggested to be involved in lipid metabo- mother to infant via breast milk and the downstream lism in the brain, was present in significantly lower con - impact of infant antiretroviral drug exposure have not centrations in the breast milk of mothers without HIV. been well described [38–41]. Additionally, there is a lack Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 9 of 18 Table 2 Baseline characteristics for the mothers and their infants selected for breast milk metabolomics N All HIV‑/HUU HIV+/HEU P value 17 17 Maternal Maternal Age (years), median (IQR) 34 32 (28, 35) 31 (26, 33) 32 (31, 37) 0.1239 Employed,n(%) 34 30 (88.2) 15 (88.2) 15 (88.2) 1 Education,n(%) 34 0.0005 Primary/junior secondary 9 (26.5) 1 (5.9) 8 (47.1) Senior secondary 10 (29.4) 3 (17.6) 7 (41.2) Tertiary 15 (44.1) 13 (76.5) 2 (11.7) Married,n(%) 34 22 (64.7) 15 (88.2) 7 (41.2) 0.0104 Parity,n(%) 34 0.4132 Primip 8 (23.5) 6 (35.3) 2 (11.8) 2–4 23 (67.7) 10 (58.8) 13 (76.4) > 4 3 (8.8) 1 (5.9) 2 (11.8) CD4 count (cells/mm ), median (IQR) 17 377 (242, 497) Baby Gender, female,n(%) 34 13 (38.2) 7 (41.2) 6 (35.3) 1 Delivery type,n(%) 34 1 Vaginal 28 (82.4) 28 (82.4) 28 (82.4) Cesarean section 6 (17.6) 3 (17.6) 3 (17.6) Premature delivery,n(%) 34 5 (15.2) 1 (6.3) 4 (23.5) 0.3353 Birth weight (Kg), median (IQR) 34 2.88 (2.5, 3.3) 3.25 (2.7, 3.5) 2.7 (2.5, 3.0) 0.0364 Birth weight <2.5 Kg,n(%) 34 6 (17.6) 2 (11.8) 4 (23.5) 0.6562 Anthropometrics at birth,z‑score, mean (SD) Weight‑for ‑age 34 − 1.05 (1.34) − 0.57 (1.41) − 1.53 (1.12) 0.0356 Meconium microbiome Shannon diversity, median (IQR) 26 1.66 (1.27, 2.18) 1.89 (1.33, 2.179) 1.32 (1.01, 2.26) 0.1134 Exclusive breastfeeding (6 months),n(%) 34 30 (88.2) 15 (88.2) 15 (88.2) 1 Breastfeeding duration (months), median (IQR) 34 9 (6, 10) 10 (9, 15) 6 (6, 9) < .0001 Trimethoprim‑sulfamethoxazole use (6 months), n(%) 34 19 (55.9) 3 (17.7) 16 (94.1) < .0001 Antibiotic use (6 months),n(%) 34 25 (73.5) 8 (47.1) 17 (100) 0.0009 W F T Wilcoxon’s, Fisher’s, t test HUU, HIV unexposed uninfected; HEU, HIV exposed uninfected; IQR, interquartile range; N, number of participants; SD, standard deviation of knowledge regarding the effect of antiretroviral drugs infected with HIV. Consequently, it is estimated that 15% ingested via breast milk have on the infants’ gut microbiota. of all infants born in sub-Saharan Africa are HEU [42]. To explore whether lamivudine and/or nevirapine in breast Although not infected with HIV, this population remains milk are associated with the relative abundance of any bac- at risk for early-life developmental abnormalities such as growth faltering [43, 44], increased morbidities [45–47], terial taxa in the infants’ gut microbiota, we performed infant diarrhea [48, 49], and higher mortality during logistic regressions. The results suggested that high nevi - the first 12––24 months of life when compared to HUU rapine concentrations correlate with significantly lower rel - infants [50–52]. ative abundances of Bifidobacterium longum in our cohort Many of the mothers with HIV in this study opted of 17 HEU infants (P = 0.040), consistent with our observa- not to breastfeed for long durations due to concerns tion that breastfeeding infants born to mothers with HIV for transmission of HIV to their babies (Fig. 2). Because exhibit significantly less Bifidobacteria at 6 months post - breastfeeding is known to have a profound impact on the partum than those born to mothers without HIV. infant gut microbiota, our statistical analyses accounted for such differential feeding practices (newborn, breast Discussion - In sub-Saharan Africa, the scale-up of antiretroviral feeding, or not breastfeeding). The relative abundances prophylaxis to prevent mother-to-child HIV transmis- of several bacterial taxa in the infant gut significantly sion has dramatically reduced the number of children differed based on breastfeeding status, one of which Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 10 of 18 Fig. 5 Heatmap of the 20 metabolites with the highest Hotelling’s T value. Heatmap of changes in breast milk metabolites at 6 weeks (purple) and 6 months (yellow) postpartum between mothers with (red) and without HIV (blue). The heatmap was created using the statistical package in MetaboAnalyst 5.0 (http:// www. metab oanal yst. ca/ Metab oAnal yst/). The heatmap is a visualization of the changes in abundance/level of breast milk metabolites (rows) for each mother (columns). The color ranges from dark red (high abundance or level) to dark blue (low abundance or level); white is no change was Bifidobacterium, that was positively associated other adverse postnatal outcomes that may, in part, reflect with breastfeeding. Early Bifidobacterium colonization the toxicity of nucleoside analogs used in ART [66, 67]. of the infant gut is facilitated by the commencement of There are limited data reporting the concentrations of breastfeeding, a phenomenon that has been described in ART drugs in nursing infants as a result of transfer via a number of other studies [24, 33, 53]. Members of the breast milk [68, 69]; however, it has been shown that genus Bifidobacterium play an important role in the mat - lamivudine and nevirapine [70, 71] are transferred to uration of the infant gut by providing butyrate-producing infants via breast milk in biologically significant concen - colonic bacteria with exogenous acetate that can be used trations [40]. Our breast milk metabolomics data showed as a co-substrate to produce butyrate, a compound with that both lamivudine and nevirapine were present in the anti-inflammatory properties that enhances intestinal breast milk of our cohort of mothers with HIV and we barrier function and mucosal immunity [54–56]. Addi- observed that significantly lower levels of Bifidobacte - tionally, Bifidobacteria have been shown to metabolize rium longum in the HEU infant gut microbiota correlated tryptophan into indole-3-lactic acid and other beneficial with high nevirapine concentration. However, there was metabolites in the infant gut [57]. no direct link between high ART drug concentrations Infants born to mothers with HIV exhibited lower and low WAZ, suggesting other factors could also be WAZ from birth throughout the 18-month study period responsible for the adverse growth outcomes. in comparison to HUU infants (Table 1, Fig. 4A). Our Besides the presence of ART drugs, we observed addi- finding of lower WAZ at birth among HEU infants is tional differences in the breast milk from mothers with consistent with several other studies conducted in Afri- and without HIV. Mothers with HIV had a significantly can populations [27, 30, 58–60]. When our HEU infants lower level of tiglyl carnitine (C5), an acylcarnitine, in were grouped by breastfeeding status, the breastfeeding their breast milk. Carnitine is involved in β-oxidation of HEU infants had significantly higher WAZ at 6 weeks fatty acids and plays other important roles in metabolism postpartum compared to non-breastfeeding HEU infants [72]. Carnitine deficiency has previously been reported (Fig. 4B). This result suggests that breastfeeding may be to occur in adult patients with HIV [73, 74] and children partially mitigating the adverse effects of maternal HIV [75], and may reflect gastrointestinal malabsorption. status; however, this benefit did not persist as the differ - Acylcarnitine is present in relatively high levels in the ence in WAZ is no longer significant at 6 months post - brain [76] and can readily cross the blood–brain barrier partum when most of the mothers with HIV had stopped [77]. Supplementation with acylcarnitine in neurological breastfeeding. diseases [78] has been shown to be of benefit by enhanc - A few studies have suggested that fetal growth may be ing lipid synthesis, altering and stabilizing membrane affected by in utero antiretroviral therapy (ART) drug composition, modulating genes and proteins, improving exposure [28, 30, 60, 61] and that ART drugs are associ- mitochondrial function, increasing antioxidant activity, ated with preterm birth and low birth weight [62–65] and and enhancing cholinergic neurotransmission [79–82]. Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 11 of 18 Another metabolite that was found at significantly gut microbiome and maturation of the immune system. higher levels in the breast milk of mothers with HIV was We were not able to determine whether the association kynurenine. Kynurenine is a major metabolite of tryp- between ART drug metabolites and Bifidobacterium lev - tophan (TRP), an essential amino acid that can only be els is a direct one, or an indirect one in which ART drug acquired through diet in humans [83]. About 99% of TRP metabolites serve as markers of HIV infection. There - is metabolized via the kynurenine pathway (KP) [84], fore, a future study to evaluate the impact of ART drug which contains several neuroactive metabolites that may metabolites in the breast milk of mothers with HIV may influence brain function in health and disease [37], and it be indicated to better understand their effects on the has been shown that the KP of TRP catabolism remains maturation of the infant gut microbiota and/or immune abnormally high in individuals with HIV [85, 86]. activation markers. Given the lower relative abundance Because overexpression of this pathway has been associ- of Bifidobacterium in HEU infants, the use of a Bifidobac - ated with adaptive immune defects, it has been shown to terium probiotic supplement may be of benefit in these have deleterious effects on disease progression and neu - populations [101]; however, this approach should be rocognition in patients with HIV. evaluated in a follow-up clinical trial targeting the critical A major strength of this study is the longitudinal window of the first 6 months postpartum. approach and large sample size. Two hundred seventy- One of the hypotheses at the start of this study was that two mother-infant-pairs were followed prenatally to we would uncover differences in the maternal microbiota 18 months postpartum (seven time points). Our results related to HIV status. Therefore, it was somewhat unex - confirm data from earlier published reports that mater - pected that we did not observe significant differences in nal HIV infection is associated with adverse growth out- the vaginal and gut microbiota between mothers with comes of HEU infants [8, 9, 52, 58, 87–99]. Moreover, and without HIV, although a similar finding was previ - our data reveal that these differences persist from birth ously reported by Bender et al. [8]. As was proposed by through at least 18 months, which is a critical window Bender et al. [8], it may be that because of the HIV proph- for the development and activation of the immune and ylaxis and/or prenatal care that was provided throughout central nervous systems [100]. Our results suggest that the study, mothers may have been “too healthy” for any the interaction between maternal HIV status, the infant potential differences in the microbiota to be significant. gut microbiota, breastfeeding, and growth outcome is The “healthfulness” of the mothers with HIV could have complex. One of the most important observations in this biased our results towards the null (i.e., no difference study is that HEU infants exhibit growth deficits over the between mothers with and without HIV), which would first 18 months of life when compared to HUU infants. mean the discovery of fewer differences that might exist While breastfeeding was shown to be of benefit to the in mothers with HIV that are not on ART therapy. HEU infants in this study, breastfed HEU infants still Nevertheless, there remains an urgent need to address exhibited lower WAZ than the HUU infants. Unfortu- the increased morbidity and mortality in HEU infants nately, the duration of breastfeeding by the HIV-positive in the months following birth that have been described mothers was relatively short, and we could not evaluate in numerous studies [8, 52, 58, 87–99]. While our data the potential impact of longer-term breastfeeding in this revealed a limited number of specific differences in the cohort. We also observed that the relative abundance of gut microbiota of HEU and HUU infants, they may be Bifidobacterium spp. was significantly lower in the gut very important with respect to growth and development microbiota of all HEU infants over the first 6 months in the first 6 months of life. Another factor to consider in postpartum, even if the HEU infants were receiving future studies is environmental enteropathy (or environ- breast milk. mental enteric dysfunction), which affects predominantly While our data provide a new level of understanding children in low-income countries and is hypothesized to of the impact of maternal HIV infection and the poten- be caused by continuous exposure to fecal contamination tial role of the gut microbiota on infant health, additional in food, water, and fomites [102]. u Th s, multiple addi - follow-up studies are needed before any practical recom- tional efforts to strengthen the maternal and infant gut, mendations can be considered. Identification of the safest including strategies to prevent or treat enteropathogen ART regimens for use in pregnancy that optimize both infections, should be a priority. maternal and child outcomes still represents a key pub- lic health challenge. Breastfeeding was of benefit in our Conclusions HEU infant cohort in the first weeks postpartum, how - Two hundred seventy-two mother-infant-pairs were fol- ever, ART drug metabolites in this cohort were associ- lowed prenatally to 18 months postpartum (seven time ated with a lower abundance of Bifidobacterium, a genus points). Our results confirm that maternal HIV infec - that is essential in the development of a healthy infant tion is associated with adverse growth outcomes of HEU Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 12 of 18 Infant HIV testing infants. Moreover, our data reveal that these differences HIV DNA PCR test was done for all HEU babies at 6 persist from birth through at least 18 months, which is weeks postpartum and at 4 months for non-breastfed a critical window for the development and activation of infants or 2 months after breastfeeding cessation. the immune and central nervous systems. Our results suggest that the interaction between maternal HIV status, the infant gut microbiota, breastfeeding, and PMTCT antiretroviral regimen growth outcome is complex. We observed that the rela- About 70% of the mothers with HIV were already on tive abundance of Bifidobacterium spp. was significantly highly active antiretroviral treatment (HAART) prior lower in the gut microbiota of all HEU infants over the to the index pregnancy, and their triple regimens were first 6 months postpartum, even if the HEU infants were continued. Others were initiated on antiretroviral drugs receiving breast milk. Breastfeeding was of benefit in in line with Nigerian guidelines, which recommend our HEU infant cohort in the first weeks postpartum, HAART for women requiring treatment for their own however, ART drug metabolites in this cohort were disease or option B prophylaxis with triple regimen until associated with a lower abundance of Bifidobacterium, 1 week after breastfeeding ceases, as well as nevirapine to a genus that is essential in the development of a healthy the baby from birth to 6 weeks. infant gut microbiome and maturation of the immune system. Evaluating the impact of ART drug metabolites in the breast milk of mothers with HIV is necessary to Clinical assessment better understand their effects on the maturation of the Standardized questionnaires were utilized at each study infant gut microbiota and/or immune activation mark- visit to document general medical and obstetric informa- ers. The use of a Bifidobacterium probiotic supplement tion, including medication and comorbidity history, gen- may be of benefit in these populations. Multiple addi - eral physical examination findings, and anthropometric tional efforts to strengthen the maternal and infant gut, assessment. including strategies to prevent or treat enteropathogen infections, should be a priority. Feeding practice Information on feeding practices was collected using Methods structured feeding questionnaires. This included type, Study design, participant visit, and data collection pattern, and duration of breastfeeding as well as comple- Design mentary and alternate feeding practices. This was a prospective cohort study of mother-infant pairs conducted at the University of Benin Teaching Hos- pital Nigeria (UBTH) between 2015 and 2018. The study Anthropometric assessment was approved by the UBTH research ethics committee Weight was measured to the nearest 0.1 Kg using “Salter and the University of Maryland Baltimore Institutional Baby Scale (Model 180)” at birth and “Seca Digital Scale Review Board. (Model 872)” subsequently. For the latter, baby’s weight was determined from the combined mother-baby weight Study participants measurement. Recumbent length was measured using Pregnant women with and without HIV infection (~ 150 an infantometer (“Seca 416”). A flexible non-elastic tape each) were recruited from the University of Benin Teach- (“Seca 212”) was used to measure head and arm circum- ing Hospital located in Edo State, Southern Nigeria. Par- ference. Low birth weight was defined as birth weight < ticipating women were required to be aged between 18 2.5 kg [103]. World Health Organization (WHO) child and 45 years, have documented evidence of HIV status, growth standards were used to generate z scores for and willing to comply with follow-up assessment sched- weight for age (WAZ). WAZ ≤ 2 z-scores were defined as ule. Babies born to these women were also enrolled underweight [104, 105]. at birth. Recruited mother-infant pairs were assessed at birth and followed up for 18 months with scheduled Sample collection assessment visits at 1, 6, 9, 15, and 18 months. Demo- Meconium and stool graphic, clinical, feeding, anthropometric and microbi- About 0.5 g of meconium and stool samples were col- ome data were collected at each visit. Informed consent lected at birth and at each follow-up study visit respec- was also obtained from all mothers. University of Mary- tively (Table S1). Similarly, 0.5 g of stool sample was land Baltimore and UBTH Institutional Review Boards collected from the mothers at enrollment and following approved all study procedures. delivery (Table S1). Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 13 of 18 Breast milk the RDP Naïve Bayesian Classifier [109] trained with the Breast milk was collected by trained research nurses at 6 SILVA v128 16S rRNA gene database [110]. ASVs of major weeks and 6 months postpartum (Table S1). After washing stool taxa were assigned species-level taxonomy using hands with soap and water and cleaning the nipples and are- speciateIT (http:// ravel- lab. org/ speci ateit). Negative con- olar area with cotton wool soaked in normal saline, 10 ml of trols generated a negligible amount of sequencing reads, breast milk was manually expressed and collected into a fal- whereas the positive controls generated the expected con tube. This was aliquoted into cryogenic vials and imme - mock community [106]. Taxa present at a relative abun- -5 diately stored at − 20 °C and later in − 80 °C freezers. dance of less than 10 across all samples was removed from the dataset. The phyloseq R package [111] was used Vaginal swab for analysis of the microbial community data. Vaginal swab was collected from the mothers at enroll- ment and again following delivery (Table S1). Specimen Sample preparation and ultra‑high‑performance liquid was collected using “Isohelix Sk-2” swab (Geneflow, Ltd, chromatography/mass spectrometry/mass spectrometry UK) following aseptic procedures. The swab was then A selection of 17 breast milk samples from mothers with inserted back into its container tube, the cap closed, and HIV and 17 breast milk samples from mothers without HIV tube placed in a ziploc with ice pack, and this was subse- at visits 6 weeks and 6 months were shipped to Metabolon, quently stored at − 70 °C freezers (Table S1). Inc. (Durham, NC, USA) for metabolomics. The samples were chosen haphazardly, making sure the baseline charac- DNA extraction and 16S rRNA gene sequencing teristics of the mothers were similar and their infants were DNA was extracted from each fecal, meconium, and vagi- breastfed for at least 6 months postpartum (Table 2). Untar- nal specimen. Both positive and negative controls (Zymo, geted metabolite profiling was carried out by Metabolon Irvine, CA) were included in the DNA extraction process Inc. (Durham, NC, USA) using ultra-high-performance and the 16S rRNA gene sequence amplification process as liquid chromatography/mass spectrometry/mass spec- previously described [106]. Samples were thawed at 4°C trometry (UHPLC/MS/MS). Breast milk was mixed with and, in aliquots of 0.15 g per tube, resuspended in 1 ml of methanol to recover chemically diverse metabolites after 1 × phosphate-buffered saline. Cell lysis was initiated with precipitating proteins. The methanol extract was divided two enzymatic incubations: 1. using 5 μl of lysozyme (10 into five fractions: two for analysis by two separate reverse- mg/ml; Amresco, Solon, OH), 13 μl of mutanolysin (11.7 U/ phase (RP) UPLC/MS/MS methods with positive ion mode μl; Sigma-Aldrich), and 3 μl of lysostaphin (4.5 U/μl; Sigma- electrospray ionization (ESI), one for analysis by RP/UPLC/ Aldrich) for an incubation of 30 min at 37 °C and, 2. using MS/MS with negative ion mode ESI, one for analysis by 10 μl of proteinase K (20 mg/ml; Research Products Interna- hydrophilic interaction (HILIC) UPLC/MS/MS with nega- tional, Mt. Prospect, IL), 50 μl of 10% SDS, and 2 μl of RNase tive ion mode ESI, and one sample was reserved for backup. (10 mg/ml) for an incubation of 45 min at 56 °C. After the The mass spectrometry (MS) analysis alternated between enzyme treatments, cells were disrupted by bead beating MS and data-dependent MS scans using dynamic exclu- in tubes with lysing matrix B (0.1-mm silica spheres; MP sion. A pooled sample was created by taking a small aliquot Biomedicals, Solon, OH), at 6 m/s at room temperature in from each of the samples, which served as technical repli- a FastPrep-24 (MP Biomedicals). The resulting crude lysate cates in the assay, whereas pure water samples served as a was processed using the ZR fecal DNA miniprep kit (Zymo, process blank, and a cocktail of quality control (QC) stand- Irvine, CA) according to the manufacturer’s recommen- ards (Metabolon) was spiked into every standard sample to dations. The samples were eluted with 100 μl of ultrapure identify the instrument variability. The instrument variabil - water into separate tubes. DNA concentrations in the sam- ity determined by calculating the median relative standard ples was determined with the Bioanalyzer 2100 DNA 1000 deviation for the internal standards was 3%. The samples chip (Agilent, Santa Clara, CA). were randomized across the platforms, and internal stand- ards and process blanks were added to each sample prior to 16S rRNA gene sequence analysis injection into the mass spectrometers. Hypervariable regions V3 and V4 of the bacterial 16S rRNA gene were amplified with primers 319F and 806R as Metabolomics data extraction, compound identification, previously described by [107, 108]. High-quality amplicon and quantification sequences were obtained on an Illumina HiSeq 2500 mod- The raw data extraction, peak identification, and QC pro - ified to generate 300 bp paired-end reads [108]. A total of cess were performed using Metabolon’s proprietary hard- 139 million reads were retained following chimera removal ware and software. The metabolites were identified using a and 45,556 amplicon sequence variants (ASVs) were gen- proprietary in-house library based on standards that con- erated by DADA2 and taxonomically classified using tained the retention time/index, mass to charge ratio, and Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 14 of 18 chromatographic data (including MS/MS spectral data) on antibiotic use, and breastfeeding at the time of visit. Addi- molecules present in the library. Additional mass spectral tionally, MaAsLin2 parameters for taxa analysis were set entries were created for structurally unnamed biochemi- as follows: P value control for Benjamini-Hochberg FDR cals, which were identified by their recurrent nature (both was set at level 0.05, the minimum abundance for each chromatographic and mass spectral). Peaks were quantified taxon was set to 1% and the minimum percent of samples using the area under the curve. The biochemical data were for which a taxon is detected at 1% was set to 10%. The normalized for the volume of breast milk used. parameters for the metabolomic analysis were as follows: Raw data was extracted, peak-identified and QC pro - P value control for Benjamini-Hochberg FDR was set at cessed using Metabolon’s hardware and software. Com- level 0.05, the minimum abundance for each metabolite pounds were identified by comparison to library entries of was set to 0.001, and the minimum percent of samples purified standards or recurrent unknown entities. Metabo - for which a metabolite is detected at 0.001 was set to 10%. lon maintains a library based on authenticated standards The heatmap was created using the statistical package in that contains the retention time/index, mass to charge ratio, MetaboAnalyst 5.0 (http:// www. metab oanal yst. ca/ Metab and chromatographic data (including MS/MS spectral data) oAnal yst/). ANOVA and post hoc test were performed by on all molecules present in the library. More than 3300 com- MetaboAnalyst 5.0. The P value was obtained by running mercially available purified standard compounds have been the Fishers’ LSD after the ANOVA test and adjusted by acquired and registered into Metabolon’s system for analysis multiple test corrections using the Benjamin-Hochberg on all platforms for determination of their analytical charac- procedure (FDR was set at level 0.05). Pearson’s correla- teristics. Additional mass spectral entries have been created tions were run using Benjamini-Hochberg multiple com- for structurally unnamed biochemicals, which have been parison adjustment (FDR P value adjustment set at 0.05). identified by virtue of their recurrent nature (both chroma - tographic and mass spectral). A data normalization step was Abbreviations performed to correct variation resulting from instrument ART : Antiretroviral therapy; HEU: HIV‑ exposed uninfected; HUU: HIV‑unex ‑ inter-day tuning differences. Essentially, each compound posed uninfected; IMC: Infant meconium; IST: Infant stool; IOS: Infant oral swab; MST: Maternal stool; MVS: Maternal vaginal swab; MBM: Maternal breast was corrected in run-day blocks by registering the medians milk; PCoA: Principal coordinates analysis; PERMANOVA: Permutational multi‑ to equal one (1.00) and normalizing each data point propor- variate analysis of variance; ASV: Amplicon sequence variant; ANOVA: Analysis tionately (termed the “block correction”). of variance; FDR: False discovery rate; UPLC: Ultra‑performance liquid chroma‑ tography; SI: Shannon index; MaAsLin2: Multivariable association with linear models; WAZ: Weight‑for ‑age z‑score; TMP ‑SMX: Trimethoprim‑sulfameth‑ Statistical analysis oxazole; TRP: Tryptophan; KP: Kynurenine pathway; OPLS‑DA: Orthogonal partial least square discriminant analysis; PCA: Principal component analysis; Statistical analyses were performed using R (version 3.6.0). WHO: World Health Organization; MEBA: Multivariate empirical Bayes analysis; Demographic and clinical characteristics were compared UHPLC/MS/MS: Ultra‑high‑performance liquid chromatography/mass spec‑ between HEU and HUU children and their mothers using trometry/mass spectrometry; RP: Reverse‑phase; ESI: Electrospray ionization; MS: Mass spectrometry; QC: Quality control; HILIC: Hydrophilic interaction. Wilcoxon, Fisher’s exact and t tests. Longitudinal compar- isons of alpha diversity were performed using univariable Supplementary Information and multivariable linear regression. Pairwise compari- The online version contains supplementary material available at https:// doi. sons were performed with post hoc Tukey HSD test with org/ 10. 1186/ s40168‑ 022‑ 01230‑1. FDR P value adjustment set at level 0.05. Principal coor- dinates analysis (PCoA) using Bray-Curtis dissimilarity Additional file 1: Figure S1. Microbiota of 272 mother ‑infant pairs. was performed to assess the beta diversity. Permutational Figure S2. Shannon diversity of 272 mother‑infant pairs. Figure S3. multivariate analysis of variance (PERMANOVA) was PCoA of maternal microbiota based on HIV status. Figure S4. Shannon diversity of maternal microbiota based on HIV status. Figure S5. Maternal conducted to test whether the bacterial communities microbiota composition showed no significant differences based on sequenced have different centroids based on HIV-status HIV status. Figure S6. Number of breastfeeding and non‑breastfeeding (mothers) or HIV-exposure (infants). Significance of the infants throughout the 18‑month study period. Figure S7. PCoA of infant microbiomes based on HIV exposure, breastfeeding status, and time point. results was confirmed with a test of heterogeneity (ensure Figure S8. PCoA of infant microbiomes based on Bifidobacterium longum homogenous dispersion). In addition, multivariate asso- relative abundance, breastfeeding status, and time point. Figure S9. Shan‑ ciation with linear models (MaAsLin2) [112], an addi- non diversity of infant gut microbiota based on HIV exposure, breastfeed‑ ing status, and time point. Figure S10. Orthogonal partial least square tive general linear model with boosting that can capture discriminant analysis (OPLS_DA) and principal component analysis (PCA) the effects of a parameter of interest while deconfound - of breast milk. Figure S11. Orthogonal partial least square discriminant ing the effects of other metadata, was used to efficiently analysis (OPLS_DA) and principal component analysis (PCA) of breast milk at six weeks postpartum and 6 months postpartum. Figure S12. Top 20 determine multivariable association between clinical metabolites with highest Hotelling’s T value. Table S1. Sample collection metadata, 16S rRNA gene sequence data, and breast milk schedule. Table S2. Two‑ way repeated measures analysis of variance metabolomic data. MaAsLin2 analysis for the infants (ANOVA) of breast milk metabolites. was adjusted for delivery type, prematurity, timepoint, Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 15 of 18 and uninfected, 2000‑18: a modelling study. Articles Lancet Glob Acknowledgements Health. 2020;8:67–75. The authors would like to thank the Genomics Resource Center at the 3. Mcgrath CJ, Nduati R, Richardson BA, Kristal AR, Mbori‑Ngacha D, Institute for Genome Sciences (University of Maryland School of Medicine) Farquhar C, et al. The prevalence of stunting is high in HIV‑1‑ exposed for the 16S rRNA amplicon sequencing, as well as gratefully acknowledge uninfected infants in Kenya 1‑3. J Nutr. 2012;142:757–63. the work of the Microbiome Service Lab and Jonathan Lim with running 4. Marinda E, Humphrey JH, Iliff PJ, Mutasa K, Nathoo KJ, Piwoz EG, et al. the sequencing data through their bioinformatics pipeline. Additionally, the Child mortality according to maternal and infant HIV status in Zimba‑ authors would like to acknowledge the laboratory staff at the University of bwe. Pediatr Infect Dis J. 2007;26:519–26. Benin Teaching Hospital and Asokoro General Hospital for collecting and 5. Newell ML, Coovadia H, Cortina‑Borja M, Rollins N, Gaillard P, Dabis F. processing of the samples. Mortality of infected and uninfected infants born to HIV‑infected moth‑ ers in Africa: a pooled analysis. Lancet. 2004;364:1236–43. Authors’ contributions 6. Slogrove A, Reikie B, Naidoo S, De Beer C, Ho K, Cotton M, et al. HIV‑ NN, CMF, MC were responsible for the conception and/or design of the study. exposed uninfected infants are at increased risk for severe infections in SG, JJ, NN, OM, AO, AS were responsible for the acquisition and/or analysis the first year of life. J Trop Pediatr. 2012;58:505–8. of clinical data. SG, OAM, CMF were responsible for the acquisition and/or 7. Wei R, Msamanga GI, Spiegelman D, Hertzmark E, Baylin A, Manji K, et al. analysis of the metabolomics data. SG, OAM, CMF were responsible for the Association between low birth weight and infant mortality in children acquisition and/or analysis of microbiome data. SG, JJ, CMF, MC conducted born to human immunodeficiency virus 1‑infected mothers in Tanza‑ the statistical analyses. All authors participated in drafting and revising the nia. Pediatr Infect Dis J. 2004;23:530–5. manuscript and have read and approved the final manuscript. 8. Bender JM, Li F, Martelly S, Byrt E, Rouzier V, Leo M, Tobin N, Pannaraj PS, Adisetiyo H, Rollie A. Maternal HIV infection influences the microbiome Funding of HIV‑uninfected infants. Sci Transl Med. 2016;8:349ra100. The research reported in this publication was supported by the National 9. Machiavelli A, Duarte RTD, de Souza Pires MM, Zárate‑Bladés CR, Pinto Institute of Dental and Craniofacial Research of the National Institutes of AR. The impact of in utero HIV exposure on gut microbiota, inflamma‑ Health under Award Number R01DE025174. The content is solely the responsi‑ tion, and microbial translocation. Gut Microbes. 2019;10:599–614. bility of the authors and does not necessarily represent the official views of 10. González R, Mandomando I, Fumadó V, Sacoor C, Macete E, Alonso PL, the National Institutes of Health. Additional support was provided by the et al., editors. Breast milk and gut microbiota in African mothers and Dean’s Endowed Professorship to Claire M. Fraser, PhD (University of Maryland infants from an area of high HIV prevalence. PLoS One. 2013;8:e80299. School of Medicine). Olivia A. Martin is a TL1 post‑ doctoral fellow supported https:// doi. org/ 10. 1371/ journ al. pone. 00802 99. by 1TL1TR003100‑01, 1UL1TR003098‑01, and the University of Maryland, 11. Prendergast AJ, Chasekwa B, Rukobo S, Govha M, Mutasa K, Ntozini Baltimore, Institute for Clinical and Translational Research. R, et al. 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Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2022
eISSN: 2049-2618
DOI: 10.1186/s40168-022-01230-1
Publisher site: See Article on Publisher Site

Abstract

Background: Access to antiretroviral therapy (ART ) during pregnancy and breastfeeding for mothers with HIV has resulted in fewer children acquiring HIV peri‑ and postnatally, resulting in an increase in the number of children who are exposed to the virus but are not infected (HEU). HEU infants have an increased likelihood of childhood infections and adverse growth outcomes, as well as increased mortality compared to their HIV‑unexposed (HUU) peers. We explored potential differences in the gut microbiota in a cohort of 272 Nigerian infants born to HIV ‑positive and nega‑ tive mothers in this study during the first 18 months of life. Results: The taxonomic composition of the maternal vaginal and gut microbiota showed no significant differ ‑ ences based on HIV status, and the composition of the infant gut microbiota at birth was similar between HUU and HEU. Longitudinal taxonomic composition of the infant gut microbiota and weight‑for ‑age z‑scores ( WAZ) differed depending on access to breast milk. HEU infants displayed overall lower WAZ than HUU infants at all time points. We observed a significantly lower relative abundance of Bifidobacterium in HEU infants at 6 months postpartum. Breast milk composition also differed by time point and HIV infection status. The antiretroviral therapy drugs, lamivudine and nevirapine, as well as kynurenine, were significantly more abundant in the breast milk of mothers with HIV. Levels of tiglyl carnitine (C5) were significantly lower in the breast milk of mothers without HIV. ART drugs in the breast milk of mothers with HIV were associated with a lower relative abundance of Bifidobacterium longum. Conclusions: Maternal HIV infection was associated with adverse growth outcomes of HEU infants in this study, and these differences persist from birth through at least 18 months, which is a critical window for the development of the immune and central nervous systems. We observed that the relative abundance of Bifidobacterium spp. was signifi‑ cantly lower in the gut microbiota of all HEU infants over the first 6 months postpartum, even if HEU infants were receiving breast milk. Breastfeeding was of benefit in our HEU infant cohort in the first weeks postpartum; however, ART drug metabolites in breast milk were associated with a lower abundance of Bifidobacterium. Keywords: HIV‑ exposed infants, Gut microbiota, Breast milk metabolome, Antiretroviral therapy, Breastfeeding, Bifidobacterium, Adverse growth outcome, Weight ‑for ‑age z‑score, Acylcarnitine, Kynurenine Background Improved access to antiretroviral therapy (ART) for *Correspondence: cmfraser@som.umaryland.edu Claire M. Fraser and Man Charurat contributed equally to this work as mothers with HIV during pregnancy and breastfeeding co‑senior authors. has resulted in fewer children acquiring HIV peri- and Department of Medicine, University of Maryland School of Medicine, postnatally [1]. There has been a resultant increase in the Baltimore, MD, USA Full list of author information is available at the end of the article number of children born to mothers with HIV who are © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 2 of 18 exposed to the virus, but who are not infected (HEU). weight was significantly lower among HEU babies (P < This population is estimated to be 14.8 million children 0.001), but the proportion of those born with low birth globally; with approximately 13.2 million children living weight (< 2.5 Kg) and those born prematurely did not in sub-Saharan Africa [2]. It has been shown previously differ significantly between HEU and HUU children. that HEU infants have an increased likelihood of child- Weight-for-age z-scores (WAZ) were significantly lower hood infections and adverse growth outcomes, as well as among HEU as compared to HUU babies at birth. The increased mortality compared to their HIV unexposed median duration of breastfeeding was 9 months, and (HUU) peers [3–7]. 46% of infants were exclusively breastfed for 6 months; Exposure to HIV in utero has been shown to impact however, in HEU infants there was a significantly the gut microbiota of HEU infants [8–11], pointing to a shorter duration of breastfeeding (P < 0.001) as well as potential link between maternal HIV status, the infant lower proportion of exclusively breastfed children (P < gut microbiota, and infant health. Perturbations in the 0.001). As expected, and consistent with standard rec- infant gut microbiota have been linked with altered ommendations, the use of TMP-SMX was significantly immunity, and increased susceptibility to disease [12– greater among HEU infants at all follow-up timepoints 16]. In addition, the breast milk microbiota has been (P < 0.001). This pattern was similar for overall antibi - previously found to differ between mothers with and otic prescription until the 9-month visit, beyond which without HIV [10] and survival of HEU infants in Africa the difference between HEU and HUU was not statisti - has been associated with breast milk oligosaccharide cally significant. The variable “antibiotic use” captured composition [17]. These breast milk oligosaccharides, in any antibiotic prescribed to the participants during their turn, have been linked to the gut microbiota of HEU [8]. clinic visit. It reflected trimethoprim-sulfamethoxazole Taken together, these findings provide considerable evi - (TMP-SMX), other antibiotics, or a combination of these, dence that maternal HIV status has a profound impact on thereby enabling us to account for any antibiotic use in the acquisition and subsequent development of the infant the analyses. During the 18-month study period, none of gut microbiota [18–21]. the infants included in this analysis became HIV-positive. We set out to further investigate the relationship between in utero HIV exposure and adverse growth out- Maternal vaginal and infant gut microbiomes increased comes in HEU by conducting a longitudinal study of the in diversity over time maternal and infant microbiota of 272 Nigerian mother- High-throughput sequencing of the hypervariable infant pairs, as well as the breast milk metabolome. We regions V3 and V4 of the 16S ribosomal RNA gene was hypothesized that acquisition of an altered gut micro- used to characterize the taxonomic composition of the biota from a mother with HIV, further exacerbated by samples collected in this study. Two sample types from differences in breast milk composition between mothers the mothers (vaginal swabs; MVS and stool; MST) were with and without HIV, negatively impacts growth and collected prior to (at time of enrollment, after 12 weeks increases the risk of adverse clinical outcomes among of gestation) and at birth (number of MST samples col- HEU infants. lected at birth was lower in comparison to prenatally due to fewer specimens being produced at that time point). Results Infant meconium (IMC) was collected at birth, and Most characteristics of mother‑infant pairs were similar infant stool samples (IST) were collected at 6 weeks, 6 regardless of motherʼs HIV status months, 9 months, 15 months, and 18 months postpar- Table 1 shows the baseline characteristics of the HEU tum (Table S1). and HUU infants and their mothers. The median age of We first looked broadly at the average microbiome mothers was 32 years, similar for women with and with- composition at each sample site over all time points. out HIV (P = 0.12). Most of the mothers were employed Principal coordinates analysis (PCoA) based on Bray- (87.1%), with no significant difference between the two Curtis dissimilarity revealed a distinct clustering of the groups (P = 0.853). The mothers of the HEU children mothers’ vaginal and stool samples (PERMANOVA, P were more likely to have lower levels of education (P < = 0.001; Fig. S1A). Consistent with this observation was 0.001), less likely to be married (P < 0.001), but more the finding that the alpha diversity between these sam - likely to be multiparous (P = 0.039). While there was a ples was significantly different with MST exhibiting a slightly greater proportion of deliveries via cesarean sec- higher diversity (Shannon index; SI = 4.05 ± 0.05 SEM) tion (33.8% vs. 28.5%) among the women with HIV, this in comparison to MVS (SI = 1.85± 0.04 SEM) (Fig. S2A; did not reach statistical significance (P = 0.418). The Shannon index, P < 0.001). Vaginal microbiome diver- women with HIV had a median CD4 count of 429 cells/ sity significantly increased after birth (Fig. 1A; Shan- ml (IQR: 285–566) at enrollment. The median birth non index, P < 0.001), while maternal stool microbiome Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 3 of 18 Table 1 Baseline maternal and infant characteristics N All HIV‑/HUU HIV+/HEU 131 141 P value Maternal Maternal Age (years), median (IQR) 263 32 (29, 36) 32 (29, 35) 32 (29, 37) 0.1248 Employed,n(%) 263 229 (87.1) 110 (86.6) 119 (87.5) 0.8527 Education,n(%) 263 < .0001 Primary/junior secondary 47 (17.9) 3 (2.4) 44 (32.4) Senior secondary 81 (30.8) 26 (20.5) 55 (40.4) Tertiary 135 (51.3) 98 (77.2) 37 (27.2) Married,n(%) 263 213 (81.0) 124 (97.6) 89 (65.4) < .0001 Parity,n(%) 262 0.0388 Primip 74 (28.2) 44 (34.9) 30 (22.1) 2–4 181 (69.1) 80 (63.5) 101 (74.3) > 4 7 (2.7) 2 (1.6) 5 (3.7) CD4 Count (cells/mm ), median (IQR) 136 428.5 (285, 566) Baby Gender, female,n(%) 272 118 (43.5) 60 (45.8) 58 (41.4) 0.4496 Delivery type,n(%) 272 0.4183 Vaginal 186 (68.4) 92 (70.2) 94 (66.7) Cesarean section 86 (31.6) 39 (29.8) 47 (33.3) Premature delivery,n(%) 268 20 (7.5) 7 (5.4) 13 (9.4) 0.2486 Birth weight (Kg), median (IQR) 271 3 (2.7, 3.4) 3.2 (2.8, 3.5) 2.9 (2.5, 3.25) < .0001 Birth weight <2.5 Kg,n(%) 271 33 (12.2) 12 (9.2) 21 (15.0) 0.1793 Anthropometrics at birth,z‑score, mean (SD) Weight‑for ‑age 271 − 0.71 (1.2) 0.39 (1.2) − 1.01 (1.2) < .0001 Meconium microbiome Shannon diversity, median (IQR) 176 1.75 (1.2, 2.3) 1.71 (1.2, 2.2) 1.78 (1.1, 2.3) 0.7386 Exclusive breastfeeding (6 months),n(%) 246 112 (45.5) 72 (62.1) 40 (30.8) < .0001 Breastfeeding duration (months), median (IQR) 272 6 (1, 9) 9 (9, 15) 1 (1, 6) < .0001 Trimethoprim‑sulfamethoxazole use (6 months),n(%) 246 131 (53.3) 15 (12.9) 116 (89.2) < .0001 Antibiotic use (6 months),n(%) 257 111 (43.2) 35 (29.7) 76 (54.7) < .0001 W F T Wilcoxon’s, Fisher’s, t test HUU, HIV‑unexposed uninfected; HEU, HIV ‑ exposed uninfected; IQR, interquartile range; N, number of participants; SD, standard deviation diversity was unchanged over this same time period between mothers with and without HIV for either of the (Fig. 1A; Shannon index, P = 0.138). sample sites (PERMANOVA, P = 0.876; Fig. S3). There The two sample types for the infants also displayed sig - were also no significant differences in bacterial diver - nificant differences (Fig. S1B). In the aggregate, IMC had sity identified based on HIV infection (Fig. S4; Shannon a lower diversity, (SI = 1.92 ± 0.05 SEM) compared to index, P = 0.672). IST (SI = 2.89 ± 0.02 SEM) (Fig. S2B; Shannon index, P < Longitudinal changes in the microbiota of mothers 0.001). As previously reported, we observed a significant with and without HIV were also investigated. No sig- increase in the diversity of IST from birth to 18 months nificant differences in the stool or vaginal microbiota postpartum (Fig. 1B; Shannon index, P < 0.001). between mothers with and without HIV (Fig. S5A) were found. The maternal stool microbiota contained both Maternal microbiome composition showed no significant Bacteroides and Prevotella from the phylum Bacteroi- differences based on HIV status detes, several genera in the Firmicutes phylum includ- To determine the impact of HIV infection on the mater- ing Blautia, Faecalibacterium, Lactobacillus, Roseburia, nal microbiota, the taxonomic composition of the micro- Staphylococcus and Streptococcus, and Bifidobacterium. biota from mothers with HIV was compared to that from The maternal vaginal microbiota was dominated by Lac - mothers without HIV. PcoA revealed no clear separation tobacillus, along with Gardnerella and Pseudomonas; Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 4 of 18 Fig. 1 Alpha diversity (Shannon index) for each sample type and timepoint. A Maternal stool (MST ) microbiome diversity did not change prenatally to birth (Shannon index, P = 0.138), whereas maternal vaginal (MSV ) microbiome diversity significantly increased after birth (Shannon index, P < 0.001). B Infant meconium (IMC) and infant stool (IST ) microbiome diversity displayed significant increases from birth to 18 months postpartum (Shannon index, P < 0.001). Horizontal lines in boxplots indicate median; boxes show first and third quartiles however, in mothers with HIV, the abundance of Lac- breast milk at these timepoints. As a result, the cohort tobacillus was lower (prenatal; 63% vs. 72%, birth; 51% was stratified not just by HIV exposure status, but also by vs. 55%) and the abundance of Gardnerella was higher breastfeeding status. (prenatal; 17% vs. 11%, birth; 13% vs. 12%) compared to In newborn infants that had not yet commenced mothers without HIV (Fig. S5B), however, these differ - breastfeeding, meconium samples were domi- ences did not reach statistical significance. At none of the nated by Pseudomonas, Enterobacter, Klebsiella, and timepoints was the bacterial diversity significantly differ - Corynebacterium. By contrast, the taxonomic compo- ent (Shannon index, P > 0.05) between mothers with and sition of all infant stool samples collected at various without HIV for any of the sample sites (Shannon index, time points postpartum was characterized by a high MVS—prenatal; P = 0.076, MVS—birth; P = 0.461, relative abundance of Bifidobacterium, Streptococcus, MST—prenatal; P = 0.761, MST – birth; P = 0.312, Fig. and Enterobacter. The relative abundances of several S5C). bacterial taxa significantly differed based on breast- feeding status as determined by MaAsLin2; Bifido- Breastfeeding status is associated with differences bacterium (FDR; P < 0.001) and Collinsella (FDR; P in the gut microbiota of infants = 0.040) were positively associated with breastfeed- At the time of this study, the World Health Organization ing [23], whereas Faecalibacterium (FDR; P = 0.007) (WHO) recommendation for mothers with HIV was to and Streptococcus (FDR; P = 0.803) were negatively exclusively breastfeed for 6 months; introduce comple- associated with breastfeeding. PcoA of stool samples mentary feeds afterwards, while continuing to breast- obtained from HEU vs. HUU infants exhibited no clear feed for up to 24 months [22]. This recommendation separation in any of the three breastfeeding groups was most relevant in situations where ART was avail- (PERMANOVA, P = 0.459; Fig. 2A); however, the able to guarantee the best chance for HIV-free survival Shannon diversity between breastfeeding HEU (SI = for exposed infants in resource-limited settings. How- 2.33 ± 0.05 SEM) and breastfeeding HUU (SI = 2.67 ± ever, many of the mothers with HIV in this study opted 0.04 SEM) was significantly different (Fig. 2B; Shannon not to breastfeed for long durations due to concerns for index, P < 0.001). There were no significant differences transmission of HIV to their babies (Table 1, Fig. S6). At between HEU (SI = 1.97 ± 0.07 SEM) and HUU (SI = 6 and 9 months postpartum, the majority (99% and 95%, 1.88 ± 0.07 SEM) infants’ alpha diversity at birth (not respectively) of HUU infants were still being breastfed, yet breastfeeding); similarly, there was no difference whereas only 39% and 17% of HEU infants were receiving between HEU (SI = 3.19 ± 0.04 SEM) and HUU (SI = Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 5 of 18 Breastfeeding HEU infants exhibit significantly higher 3.32 ± 0.06 SEM) alpha diversity in infants that are not weight‑for‑age z‑scores compared to non‑breastfeeding breastfeeding. None of the differences in relative abun- HEU infants at 6 weeks postpartum dance of bacterial taxa reached significance between Previous studies have shown that HEU infants have lower the microbiota of HEU and HUU infants within any of WAZ compared to HUU infants [27–30]. Our results the groups based on the breastfeeding status (Fig. 2C). are also consistent with those findings (Fig. 4A), which prompted us to further investigate whether there is a potential association between breastfeeding status and Breastfeeding infants from mothers with HIV exhibit WAZ in HUU and HEU infants. We ran linear regres- significantly less Bifidobacteria at 6 months postpartum sion models, followed by a pairwise post hoc test, which Previous research has shown the importance of breast- revealed that at 6 weeks postpartum, non-breastfeeding feeding and its ability to shape the gut microbiota in HEU exhibit significantly lower WAZ (− 1.82 ± 0.20 early life, both directly by exposure of the neonate to SEM) in comparison to breastfed HEU infants (− 0.99 ± the milk microbiota and indirectly, via maternal milk 0.11 SEM) (Fig. 4B, FDR; P < 0.001). That observation was factors that affect bacterial growth and metabolism no longer significant at 6 months postpartum or any later [24–26]. Because our data demonstrated that breast time points. The comparison of breastfeeding and non- feeding is associated with significant differences in breastfeeding HUU infants did not reveal any significant the diversity and composition of the gut microbiota differences at any time point. Comparing HUU with HEU in both HEU and HUU infants [8, 10], we performed by breastfeeding status and time point demonstrated that a cross-sectional comparison of the infant gut micro- HEU infants present with overall lower WAZ than HUU biota by time point. PCoA did not reveal any signifi- infants; newborn, breastfeeding, and non-breastfeeding cantly distinct clusters based on breastfeeding status HEU infants exhibit significantly lower WAZ at all time or time postpartum, although the distribution of data points (Fig. 4A, FDR; all P < 0.001). points was more dispersed in the breastfeeding cohort The gastrointestinal tract of full-term healthy infants (PERMANOVA, all P > 0.05; Fig. S7). This heteroge- is typically dominated by the genus Bifidobacterium neity in the distribution of data points correlated with [31–33]. Then, in the first months postpartum, the loss differences in the relative abundance of Bifidobacte- of Bifidobacterium species and/or gain of other bacte - rium longum (Fig. S8). Gut microbiota samples with ria can significantly alter the maturation of the micro - low B. longum clustered to the left of the PCoA, and bial community, which may lead to a variety of negative gut microbiota samples with high B. longum content consequences for host health including a predisposition were on the right. There were no significant differ- to autoimmune and metabolic diseases [34, 35]. Our ences in the Shannon diversity between HEU and HUU data revealed a significantly lower relative abundance of infant gut microbiota at any of the time points when Bifidobacterium in HEU infants at 6 months postpar - grouped by breastfeeding status (Fig. S9). We did, tum. Since breastfeeding is known to promote Bifido - however observe that one bacterial taxon, Bifidobacte- bacterium growth in the infant gut [33, 36], we explored rium, was significantly more abundant in the breast- whether the lack of breastfeeding in HEU infants during feeding HUU infants when compared to breastfeeding the first weeks and months postpartum was associated HEU infants at 6 months postpartum (Fig. 3A; FDR; with a lower abundance of Bifidobacteria. Indeed, we did P = 0.015). This difference was no longer observed in observe significantly lower relative abundances of Bifido - samples collected at 9, 15, and 18 months postpartum, bacteria in non-breastfed HEU infants at 6 weeks (15.80% which may, in part, reflect the introduction of solid ± 3.45% SEM) and 6 months (18.1% ± 1.90% SEM) post- foods at around 6 months of age. None of the taxa partum when compared to breastfeeding HEU infants at within the non-breastfeeding cohort reached statistical six weeks (34.93% ± 1.53% SEM) and 6 months (28.12% significance between HEU and HUU infants (Fig. 3B). (See figure on next page.) Fig. 2 The gut microbiota in breastfeeding infants from mothers with HIV differs from that seen in mothers without HIV. For these analyses, data across all time points were aggregated based on maternal HIV and/or breastfeeding status. A PCoA comparing HEU to HUU infants exhibited no significant separation within breastfeeding groups (PERMANOVA, P = 0.459). B The bacterial diversity of breastfeeding HEU infants (SI = 2.33 ± 0.05 SEM) and breastfeeding HUU infants (SI = 2.67 ± 0.04 SEM) showed a significant difference (Shannon index, P < 0.001). There were no significant differences in the Shannon diversity between HEU (SI = 1.97 ± 0.07 SEM) and HUU (SI = 1.88 ± 0.07 SEM) infants at birth (newborn); similarly, there was no differences between HEU infants (SI = 3.19 ± 0.04 SEM) and HUU infants (SI = 3.32 ± 0.06 SEM) infants that are not breastfeeding. C None of the bacterial taxa relative abundances differences reached significance between the microbiota of HEU and HUU infants (only genera made up with ASVs with a mean greater than 0.5% are shown) Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 6 of 18 Fig. 2 (See legend on previous page.) Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 7 of 18 Fig. 3 Breastfeeding infants from mothers with HIV exhibit significantly less Bifidobacteria at 6 months postpartum. A Bacterial taxa relative abundance differences between HEU and HUU infants within the breastfeeding cohort: Bifidobacterium was the only taxon that significantly differed between HEU and HUU infants at 6 months postpartum (6m pp, FDR; P = 0.015). B Bacterial taxa relative abundance differences between HEU and HUU infants within the non‑breastfeeding cohort: None of the bacterial taxa relative abundance differences in the non‑breastfeeding cohort reached significance between the microbiota of HEU and HUU infants. Because the majority of infants born to mothers without HIV were breastfed for 9 months, it was not possible to compare across non‑breastfeeding cohorts at these earlier time points. Only genera representing ASVs with a mean greater than 0.5% are shown at 6 weeks and 6 months postpartum. Table 2 shows that ± 2.29% SEM) (P < 0.001 and P = 0.008, respectively; the 34 mothers were a good representation of the entire Fig. 4C). cohort, with the exception of parity that was not signifi - To determine whether a low relative abundance of Bifi - cantly different between the two groups of mothers in dobacteria in HEU infants is a predictor of low WAZ, this subset, whereas it was significantly different in the we ran a logistic regression model with breastfeed- total cohort. ing included as a covariate. The model confirmed a link To analyze potential variations between the breast milk between low Bifidobacterium abundance and low WAZ of mothers with and without HIV, supervised analysis in HEU infants (β = 0.09, P = 0.018). with orthogonal partial least square discriminant analy- sis (OPLS-DA) and unsupervised analysis with princi- Breast milk composition differed by time point and HIV pal component analysis (PCA), were performed. A clear infection status separation between breast milk collected at 6 weeks and Breast milk metabolites from a subset of exclusively breast milk collected at 6 months was seen (not separated breastfeeding mothers with and without HIV were by HIV infection; Fig. S10A), which was also observed examined in more detail. Untargeted metabolite profil - in the PCA analysis (Fig. S10B). The metabolomics data ing was carried out using ultra-high-performance liquid were also analyzed by time point to identify potential chromatography/mass spectrometry/mass spectrom- differences by HIV infection. OPLS-DA showed a sepa - etry (UHPLC/MS/MS) to characterize a wide range of ration between the breast milk of mothers with and with- metabolites (a total of 553 compounds of known iden- out HIV (Fig. S11AB), however, that separation was not tity) present in breast milk samples from 34 mothers as strong in the PCA analysis and did not reach signifi - (Table 2; 17 exclusively breastfeeding mothers with HIV cance for either of the two time points (Fig. S11CD). and 17 exclusively breastfeeding mothers without HIV) Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 8 of 18 Fig. 4 Breastfeeding HEU infants exhibit significantly higher weight ‑for ‑age z‑scores compared to non‑breastfed HEU infants at 6 weeks postpartum. A HEU infants have significantly lower weight ‑for ‑age z‑scores ( WAZ) compared to HUU infants at all time points (FDR; P < 0.001). B At 6 weeks postpartum, non‑breastfeeding HEU infants exhibit significantly lower WAZ (− 1.82 ± 0.20) in comparison to breastfed HEU infants (− 0.99 ± 0.11) (FDR; P < 0.001). C Not breastfeeding HEU infants exhibited significantly lower relative abundances of Bifidobacteria at 6 weeks (15.80% ± 3.45% SEM) and 6 months (18.1% ± 1.90% SEM) postpartum when compared to breastfeeding HEU infants at 6 weeks (34.93% ± 1.53% SEM) and 6 months (28.12% ± 2.29% SEM) (P < 0.001 and P = 0.008, respectively) Two-way repeated measures analysis of variance Multivariate empirical Bayes analysis (MEBA) was used (ANOVA) (within subject) was used to identify signifi - to compare the time-course profiles between the breast cant differences in metabolites present in the breast milk milk of mothers with and without HIV. Metabolites with of mothers with and without at 6 weeks and 6 months high Hotelling’s T values comprise those whose profiles postpartum. ANOVA identified 106 metabolites that are more different between the breast milk of moth - significantly differed between the two groups and time ers with and without HIV across the time points. The points (FDR P < 0.05; Table S2). Among the 106 metab- 20 metabolites with the highest Hotelling’s T value are olites, 16 were associated with HIV infection, 88 were represented in Fig. 5 as a heatmap. The time course pro - associated with time point (6 weeks vs. 6 months post- files of the metabolites with the highest Hotelling’s T partum), and two were associated with both HIV infec- value are shown in Fig. S12. To determine whether the tion and time point. Within the 18 metabolites that were levels of any of these 20 metabolites are associated with significantly different between the breast milk of moth - the infants’ WAZ, Pearson’s correlations were run. After ers with and without HIV, 16 were higher and two were multiple comparison adjustment (FDR P value adjust- lower in the breast milk of mothers with HIV in com- ment set at 0.05), none of the 20 metabolites exhibited a parison to breast milk from mothers without HIV (Table significant correlation with WAZ. S2). The antiretroviral therapy (ART) drugs lamivudine and nevirapine were significantly more abundant in the Antiretroviral drugs found in breast milk of mothers breast milk of mothers with HIV. Kynurenine, which with HIV are associated with a lower relative abundance has received increasing attention due to its connection of Bifidobacterium longum to inflammation, the immune system, and neurological Previous studies have shown that antiretrovirals admin- conditions [37] was also significantly more abundant in istered to nursing mothers are present in their breast breast milk from mothers with HIV. Tiglyl carnitine (C5), milk; however, the degree of antiretroviral transfer from an acylcarnitine suggested to be involved in lipid metabo- mother to infant via breast milk and the downstream lism in the brain, was present in significantly lower con - impact of infant antiretroviral drug exposure have not centrations in the breast milk of mothers without HIV. been well described [38–41]. Additionally, there is a lack Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 9 of 18 Table 2 Baseline characteristics for the mothers and their infants selected for breast milk metabolomics N All HIV‑/HUU HIV+/HEU P value 17 17 Maternal Maternal Age (years), median (IQR) 34 32 (28, 35) 31 (26, 33) 32 (31, 37) 0.1239 Employed,n(%) 34 30 (88.2) 15 (88.2) 15 (88.2) 1 Education,n(%) 34 0.0005 Primary/junior secondary 9 (26.5) 1 (5.9) 8 (47.1) Senior secondary 10 (29.4) 3 (17.6) 7 (41.2) Tertiary 15 (44.1) 13 (76.5) 2 (11.7) Married,n(%) 34 22 (64.7) 15 (88.2) 7 (41.2) 0.0104 Parity,n(%) 34 0.4132 Primip 8 (23.5) 6 (35.3) 2 (11.8) 2–4 23 (67.7) 10 (58.8) 13 (76.4) > 4 3 (8.8) 1 (5.9) 2 (11.8) CD4 count (cells/mm ), median (IQR) 17 377 (242, 497) Baby Gender, female,n(%) 34 13 (38.2) 7 (41.2) 6 (35.3) 1 Delivery type,n(%) 34 1 Vaginal 28 (82.4) 28 (82.4) 28 (82.4) Cesarean section 6 (17.6) 3 (17.6) 3 (17.6) Premature delivery,n(%) 34 5 (15.2) 1 (6.3) 4 (23.5) 0.3353 Birth weight (Kg), median (IQR) 34 2.88 (2.5, 3.3) 3.25 (2.7, 3.5) 2.7 (2.5, 3.0) 0.0364 Birth weight <2.5 Kg,n(%) 34 6 (17.6) 2 (11.8) 4 (23.5) 0.6562 Anthropometrics at birth,z‑score, mean (SD) Weight‑for ‑age 34 − 1.05 (1.34) − 0.57 (1.41) − 1.53 (1.12) 0.0356 Meconium microbiome Shannon diversity, median (IQR) 26 1.66 (1.27, 2.18) 1.89 (1.33, 2.179) 1.32 (1.01, 2.26) 0.1134 Exclusive breastfeeding (6 months),n(%) 34 30 (88.2) 15 (88.2) 15 (88.2) 1 Breastfeeding duration (months), median (IQR) 34 9 (6, 10) 10 (9, 15) 6 (6, 9) < .0001 Trimethoprim‑sulfamethoxazole use (6 months), n(%) 34 19 (55.9) 3 (17.7) 16 (94.1) < .0001 Antibiotic use (6 months),n(%) 34 25 (73.5) 8 (47.1) 17 (100) 0.0009 W F T Wilcoxon’s, Fisher’s, t test HUU, HIV unexposed uninfected; HEU, HIV exposed uninfected; IQR, interquartile range; N, number of participants; SD, standard deviation of knowledge regarding the effect of antiretroviral drugs infected with HIV. Consequently, it is estimated that 15% ingested via breast milk have on the infants’ gut microbiota. of all infants born in sub-Saharan Africa are HEU [42]. To explore whether lamivudine and/or nevirapine in breast Although not infected with HIV, this population remains milk are associated with the relative abundance of any bac- at risk for early-life developmental abnormalities such as growth faltering [43, 44], increased morbidities [45–47], terial taxa in the infants’ gut microbiota, we performed infant diarrhea [48, 49], and higher mortality during logistic regressions. The results suggested that high nevi - the first 12––24 months of life when compared to HUU rapine concentrations correlate with significantly lower rel - infants [50–52]. ative abundances of Bifidobacterium longum in our cohort Many of the mothers with HIV in this study opted of 17 HEU infants (P = 0.040), consistent with our observa- not to breastfeed for long durations due to concerns tion that breastfeeding infants born to mothers with HIV for transmission of HIV to their babies (Fig. 2). Because exhibit significantly less Bifidobacteria at 6 months post - breastfeeding is known to have a profound impact on the partum than those born to mothers without HIV. infant gut microbiota, our statistical analyses accounted for such differential feeding practices (newborn, breast Discussion - In sub-Saharan Africa, the scale-up of antiretroviral feeding, or not breastfeeding). The relative abundances prophylaxis to prevent mother-to-child HIV transmis- of several bacterial taxa in the infant gut significantly sion has dramatically reduced the number of children differed based on breastfeeding status, one of which Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 10 of 18 Fig. 5 Heatmap of the 20 metabolites with the highest Hotelling’s T value. Heatmap of changes in breast milk metabolites at 6 weeks (purple) and 6 months (yellow) postpartum between mothers with (red) and without HIV (blue). The heatmap was created using the statistical package in MetaboAnalyst 5.0 (http:// www. metab oanal yst. ca/ Metab oAnal yst/). The heatmap is a visualization of the changes in abundance/level of breast milk metabolites (rows) for each mother (columns). The color ranges from dark red (high abundance or level) to dark blue (low abundance or level); white is no change was Bifidobacterium, that was positively associated other adverse postnatal outcomes that may, in part, reflect with breastfeeding. Early Bifidobacterium colonization the toxicity of nucleoside analogs used in ART [66, 67]. of the infant gut is facilitated by the commencement of There are limited data reporting the concentrations of breastfeeding, a phenomenon that has been described in ART drugs in nursing infants as a result of transfer via a number of other studies [24, 33, 53]. Members of the breast milk [68, 69]; however, it has been shown that genus Bifidobacterium play an important role in the mat - lamivudine and nevirapine [70, 71] are transferred to uration of the infant gut by providing butyrate-producing infants via breast milk in biologically significant concen - colonic bacteria with exogenous acetate that can be used trations [40]. Our breast milk metabolomics data showed as a co-substrate to produce butyrate, a compound with that both lamivudine and nevirapine were present in the anti-inflammatory properties that enhances intestinal breast milk of our cohort of mothers with HIV and we barrier function and mucosal immunity [54–56]. Addi- observed that significantly lower levels of Bifidobacte - tionally, Bifidobacteria have been shown to metabolize rium longum in the HEU infant gut microbiota correlated tryptophan into indole-3-lactic acid and other beneficial with high nevirapine concentration. However, there was metabolites in the infant gut [57]. no direct link between high ART drug concentrations Infants born to mothers with HIV exhibited lower and low WAZ, suggesting other factors could also be WAZ from birth throughout the 18-month study period responsible for the adverse growth outcomes. in comparison to HUU infants (Table 1, Fig. 4A). Our Besides the presence of ART drugs, we observed addi- finding of lower WAZ at birth among HEU infants is tional differences in the breast milk from mothers with consistent with several other studies conducted in Afri- and without HIV. Mothers with HIV had a significantly can populations [27, 30, 58–60]. When our HEU infants lower level of tiglyl carnitine (C5), an acylcarnitine, in were grouped by breastfeeding status, the breastfeeding their breast milk. Carnitine is involved in β-oxidation of HEU infants had significantly higher WAZ at 6 weeks fatty acids and plays other important roles in metabolism postpartum compared to non-breastfeeding HEU infants [72]. Carnitine deficiency has previously been reported (Fig. 4B). This result suggests that breastfeeding may be to occur in adult patients with HIV [73, 74] and children partially mitigating the adverse effects of maternal HIV [75], and may reflect gastrointestinal malabsorption. status; however, this benefit did not persist as the differ - Acylcarnitine is present in relatively high levels in the ence in WAZ is no longer significant at 6 months post - brain [76] and can readily cross the blood–brain barrier partum when most of the mothers with HIV had stopped [77]. Supplementation with acylcarnitine in neurological breastfeeding. diseases [78] has been shown to be of benefit by enhanc - A few studies have suggested that fetal growth may be ing lipid synthesis, altering and stabilizing membrane affected by in utero antiretroviral therapy (ART) drug composition, modulating genes and proteins, improving exposure [28, 30, 60, 61] and that ART drugs are associ- mitochondrial function, increasing antioxidant activity, ated with preterm birth and low birth weight [62–65] and and enhancing cholinergic neurotransmission [79–82]. Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 11 of 18 Another metabolite that was found at significantly gut microbiome and maturation of the immune system. higher levels in the breast milk of mothers with HIV was We were not able to determine whether the association kynurenine. Kynurenine is a major metabolite of tryp- between ART drug metabolites and Bifidobacterium lev - tophan (TRP), an essential amino acid that can only be els is a direct one, or an indirect one in which ART drug acquired through diet in humans [83]. About 99% of TRP metabolites serve as markers of HIV infection. There - is metabolized via the kynurenine pathway (KP) [84], fore, a future study to evaluate the impact of ART drug which contains several neuroactive metabolites that may metabolites in the breast milk of mothers with HIV may influence brain function in health and disease [37], and it be indicated to better understand their effects on the has been shown that the KP of TRP catabolism remains maturation of the infant gut microbiota and/or immune abnormally high in individuals with HIV [85, 86]. activation markers. Given the lower relative abundance Because overexpression of this pathway has been associ- of Bifidobacterium in HEU infants, the use of a Bifidobac - ated with adaptive immune defects, it has been shown to terium probiotic supplement may be of benefit in these have deleterious effects on disease progression and neu - populations [101]; however, this approach should be rocognition in patients with HIV. evaluated in a follow-up clinical trial targeting the critical A major strength of this study is the longitudinal window of the first 6 months postpartum. approach and large sample size. Two hundred seventy- One of the hypotheses at the start of this study was that two mother-infant-pairs were followed prenatally to we would uncover differences in the maternal microbiota 18 months postpartum (seven time points). Our results related to HIV status. Therefore, it was somewhat unex - confirm data from earlier published reports that mater - pected that we did not observe significant differences in nal HIV infection is associated with adverse growth out- the vaginal and gut microbiota between mothers with comes of HEU infants [8, 9, 52, 58, 87–99]. Moreover, and without HIV, although a similar finding was previ - our data reveal that these differences persist from birth ously reported by Bender et al. [8]. As was proposed by through at least 18 months, which is a critical window Bender et al. [8], it may be that because of the HIV proph- for the development and activation of the immune and ylaxis and/or prenatal care that was provided throughout central nervous systems [100]. Our results suggest that the study, mothers may have been “too healthy” for any the interaction between maternal HIV status, the infant potential differences in the microbiota to be significant. gut microbiota, breastfeeding, and growth outcome is The “healthfulness” of the mothers with HIV could have complex. One of the most important observations in this biased our results towards the null (i.e., no difference study is that HEU infants exhibit growth deficits over the between mothers with and without HIV), which would first 18 months of life when compared to HUU infants. mean the discovery of fewer differences that might exist While breastfeeding was shown to be of benefit to the in mothers with HIV that are not on ART therapy. HEU infants in this study, breastfed HEU infants still Nevertheless, there remains an urgent need to address exhibited lower WAZ than the HUU infants. Unfortu- the increased morbidity and mortality in HEU infants nately, the duration of breastfeeding by the HIV-positive in the months following birth that have been described mothers was relatively short, and we could not evaluate in numerous studies [8, 52, 58, 87–99]. While our data the potential impact of longer-term breastfeeding in this revealed a limited number of specific differences in the cohort. We also observed that the relative abundance of gut microbiota of HEU and HUU infants, they may be Bifidobacterium spp. was significantly lower in the gut very important with respect to growth and development microbiota of all HEU infants over the first 6 months in the first 6 months of life. Another factor to consider in postpartum, even if the HEU infants were receiving future studies is environmental enteropathy (or environ- breast milk. mental enteric dysfunction), which affects predominantly While our data provide a new level of understanding children in low-income countries and is hypothesized to of the impact of maternal HIV infection and the poten- be caused by continuous exposure to fecal contamination tial role of the gut microbiota on infant health, additional in food, water, and fomites [102]. u Th s, multiple addi - follow-up studies are needed before any practical recom- tional efforts to strengthen the maternal and infant gut, mendations can be considered. Identification of the safest including strategies to prevent or treat enteropathogen ART regimens for use in pregnancy that optimize both infections, should be a priority. maternal and child outcomes still represents a key pub- lic health challenge. Breastfeeding was of benefit in our Conclusions HEU infant cohort in the first weeks postpartum, how - Two hundred seventy-two mother-infant-pairs were fol- ever, ART drug metabolites in this cohort were associ- lowed prenatally to 18 months postpartum (seven time ated with a lower abundance of Bifidobacterium, a genus points). Our results confirm that maternal HIV infec - that is essential in the development of a healthy infant tion is associated with adverse growth outcomes of HEU Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 12 of 18 Infant HIV testing infants. Moreover, our data reveal that these differences HIV DNA PCR test was done for all HEU babies at 6 persist from birth through at least 18 months, which is weeks postpartum and at 4 months for non-breastfed a critical window for the development and activation of infants or 2 months after breastfeeding cessation. the immune and central nervous systems. Our results suggest that the interaction between maternal HIV status, the infant gut microbiota, breastfeeding, and PMTCT antiretroviral regimen growth outcome is complex. We observed that the rela- About 70% of the mothers with HIV were already on tive abundance of Bifidobacterium spp. was significantly highly active antiretroviral treatment (HAART) prior lower in the gut microbiota of all HEU infants over the to the index pregnancy, and their triple regimens were first 6 months postpartum, even if the HEU infants were continued. Others were initiated on antiretroviral drugs receiving breast milk. Breastfeeding was of benefit in in line with Nigerian guidelines, which recommend our HEU infant cohort in the first weeks postpartum, HAART for women requiring treatment for their own however, ART drug metabolites in this cohort were disease or option B prophylaxis with triple regimen until associated with a lower abundance of Bifidobacterium, 1 week after breastfeeding ceases, as well as nevirapine to a genus that is essential in the development of a healthy the baby from birth to 6 weeks. infant gut microbiome and maturation of the immune system. Evaluating the impact of ART drug metabolites in the breast milk of mothers with HIV is necessary to Clinical assessment better understand their effects on the maturation of the Standardized questionnaires were utilized at each study infant gut microbiota and/or immune activation mark- visit to document general medical and obstetric informa- ers. The use of a Bifidobacterium probiotic supplement tion, including medication and comorbidity history, gen- may be of benefit in these populations. Multiple addi - eral physical examination findings, and anthropometric tional efforts to strengthen the maternal and infant gut, assessment. including strategies to prevent or treat enteropathogen infections, should be a priority. Feeding practice Information on feeding practices was collected using Methods structured feeding questionnaires. This included type, Study design, participant visit, and data collection pattern, and duration of breastfeeding as well as comple- Design mentary and alternate feeding practices. This was a prospective cohort study of mother-infant pairs conducted at the University of Benin Teaching Hos- pital Nigeria (UBTH) between 2015 and 2018. The study Anthropometric assessment was approved by the UBTH research ethics committee Weight was measured to the nearest 0.1 Kg using “Salter and the University of Maryland Baltimore Institutional Baby Scale (Model 180)” at birth and “Seca Digital Scale Review Board. (Model 872)” subsequently. For the latter, baby’s weight was determined from the combined mother-baby weight Study participants measurement. Recumbent length was measured using Pregnant women with and without HIV infection (~ 150 an infantometer (“Seca 416”). A flexible non-elastic tape each) were recruited from the University of Benin Teach- (“Seca 212”) was used to measure head and arm circum- ing Hospital located in Edo State, Southern Nigeria. Par- ference. Low birth weight was defined as birth weight < ticipating women were required to be aged between 18 2.5 kg [103]. World Health Organization (WHO) child and 45 years, have documented evidence of HIV status, growth standards were used to generate z scores for and willing to comply with follow-up assessment sched- weight for age (WAZ). WAZ ≤ 2 z-scores were defined as ule. Babies born to these women were also enrolled underweight [104, 105]. at birth. Recruited mother-infant pairs were assessed at birth and followed up for 18 months with scheduled Sample collection assessment visits at 1, 6, 9, 15, and 18 months. Demo- Meconium and stool graphic, clinical, feeding, anthropometric and microbi- About 0.5 g of meconium and stool samples were col- ome data were collected at each visit. Informed consent lected at birth and at each follow-up study visit respec- was also obtained from all mothers. University of Mary- tively (Table S1). Similarly, 0.5 g of stool sample was land Baltimore and UBTH Institutional Review Boards collected from the mothers at enrollment and following approved all study procedures. delivery (Table S1). Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 13 of 18 Breast milk the RDP Naïve Bayesian Classifier [109] trained with the Breast milk was collected by trained research nurses at 6 SILVA v128 16S rRNA gene database [110]. ASVs of major weeks and 6 months postpartum (Table S1). After washing stool taxa were assigned species-level taxonomy using hands with soap and water and cleaning the nipples and are- speciateIT (http:// ravel- lab. org/ speci ateit). Negative con- olar area with cotton wool soaked in normal saline, 10 ml of trols generated a negligible amount of sequencing reads, breast milk was manually expressed and collected into a fal- whereas the positive controls generated the expected con tube. This was aliquoted into cryogenic vials and imme - mock community [106]. Taxa present at a relative abun- -5 diately stored at − 20 °C and later in − 80 °C freezers. dance of less than 10 across all samples was removed from the dataset. The phyloseq R package [111] was used Vaginal swab for analysis of the microbial community data. Vaginal swab was collected from the mothers at enroll- ment and again following delivery (Table S1). Specimen Sample preparation and ultra‑high‑performance liquid was collected using “Isohelix Sk-2” swab (Geneflow, Ltd, chromatography/mass spectrometry/mass spectrometry UK) following aseptic procedures. The swab was then A selection of 17 breast milk samples from mothers with inserted back into its container tube, the cap closed, and HIV and 17 breast milk samples from mothers without HIV tube placed in a ziploc with ice pack, and this was subse- at visits 6 weeks and 6 months were shipped to Metabolon, quently stored at − 70 °C freezers (Table S1). Inc. (Durham, NC, USA) for metabolomics. The samples were chosen haphazardly, making sure the baseline charac- DNA extraction and 16S rRNA gene sequencing teristics of the mothers were similar and their infants were DNA was extracted from each fecal, meconium, and vagi- breastfed for at least 6 months postpartum (Table 2). Untar- nal specimen. Both positive and negative controls (Zymo, geted metabolite profiling was carried out by Metabolon Irvine, CA) were included in the DNA extraction process Inc. (Durham, NC, USA) using ultra-high-performance and the 16S rRNA gene sequence amplification process as liquid chromatography/mass spectrometry/mass spec- previously described [106]. Samples were thawed at 4°C trometry (UHPLC/MS/MS). Breast milk was mixed with and, in aliquots of 0.15 g per tube, resuspended in 1 ml of methanol to recover chemically diverse metabolites after 1 × phosphate-buffered saline. Cell lysis was initiated with precipitating proteins. The methanol extract was divided two enzymatic incubations: 1. using 5 μl of lysozyme (10 into five fractions: two for analysis by two separate reverse- mg/ml; Amresco, Solon, OH), 13 μl of mutanolysin (11.7 U/ phase (RP) UPLC/MS/MS methods with positive ion mode μl; Sigma-Aldrich), and 3 μl of lysostaphin (4.5 U/μl; Sigma- electrospray ionization (ESI), one for analysis by RP/UPLC/ Aldrich) for an incubation of 30 min at 37 °C and, 2. using MS/MS with negative ion mode ESI, one for analysis by 10 μl of proteinase K (20 mg/ml; Research Products Interna- hydrophilic interaction (HILIC) UPLC/MS/MS with nega- tional, Mt. Prospect, IL), 50 μl of 10% SDS, and 2 μl of RNase tive ion mode ESI, and one sample was reserved for backup. (10 mg/ml) for an incubation of 45 min at 56 °C. After the The mass spectrometry (MS) analysis alternated between enzyme treatments, cells were disrupted by bead beating MS and data-dependent MS scans using dynamic exclu- in tubes with lysing matrix B (0.1-mm silica spheres; MP sion. A pooled sample was created by taking a small aliquot Biomedicals, Solon, OH), at 6 m/s at room temperature in from each of the samples, which served as technical repli- a FastPrep-24 (MP Biomedicals). The resulting crude lysate cates in the assay, whereas pure water samples served as a was processed using the ZR fecal DNA miniprep kit (Zymo, process blank, and a cocktail of quality control (QC) stand- Irvine, CA) according to the manufacturer’s recommen- ards (Metabolon) was spiked into every standard sample to dations. The samples were eluted with 100 μl of ultrapure identify the instrument variability. The instrument variabil - water into separate tubes. DNA concentrations in the sam- ity determined by calculating the median relative standard ples was determined with the Bioanalyzer 2100 DNA 1000 deviation for the internal standards was 3%. The samples chip (Agilent, Santa Clara, CA). were randomized across the platforms, and internal stand- ards and process blanks were added to each sample prior to 16S rRNA gene sequence analysis injection into the mass spectrometers. Hypervariable regions V3 and V4 of the bacterial 16S rRNA gene were amplified with primers 319F and 806R as Metabolomics data extraction, compound identification, previously described by [107, 108]. High-quality amplicon and quantification sequences were obtained on an Illumina HiSeq 2500 mod- The raw data extraction, peak identification, and QC pro - ified to generate 300 bp paired-end reads [108]. A total of cess were performed using Metabolon’s proprietary hard- 139 million reads were retained following chimera removal ware and software. The metabolites were identified using a and 45,556 amplicon sequence variants (ASVs) were gen- proprietary in-house library based on standards that con- erated by DADA2 and taxonomically classified using tained the retention time/index, mass to charge ratio, and Grant‑Beurmann et al. Microbiome (2022) 10:61 Page 14 of 18 chromatographic data (including MS/MS spectral data) on antibiotic use, and breastfeeding at the time of visit. Addi- molecules present in the library. Additional mass spectral tionally, MaAsLin2 parameters for taxa analysis were set entries were created for structurally unnamed biochemi- as follows: P value control for Benjamini-Hochberg FDR cals, which were identified by their recurrent nature (both was set at level 0.05, the minimum abundance for each chromatographic and mass spectral). Peaks were quantified taxon was set to 1% and the minimum percent of samples using the area under the curve. The biochemical data were for which a taxon is detected at 1% was set to 10%. The normalized for the volume of breast milk used. parameters for the metabolomic analysis were as follows: Raw data was extracted, peak-identified and QC pro - P value control for Benjamini-Hochberg FDR was set at cessed using Metabolon’s hardware and software. Com- level 0.05, the minimum abundance for each metabolite pounds were identified by comparison to library entries of was set to 0.001, and the minimum percent of samples purified standards or recurrent unknown entities. Metabo - for which a metabolite is detected at 0.001 was set to 10%. lon maintains a library based on authenticated standards The heatmap was created using the statistical package in that contains the retention time/index, mass to charge ratio, MetaboAnalyst 5.0 (http:// www. metab oanal yst. ca/ Metab and chromatographic data (including MS/MS spectral data) oAnal yst/). ANOVA and post hoc test were performed by on all molecules present in the library. More than 3300 com- MetaboAnalyst 5.0. The P value was obtained by running mercially available purified standard compounds have been the Fishers’ LSD after the ANOVA test and adjusted by acquired and registered into Metabolon’s system for analysis multiple test corrections using the Benjamin-Hochberg on all platforms for determination of their analytical charac- procedure (FDR was set at level 0.05). Pearson’s correla- teristics. Additional mass spectral entries have been created tions were run using Benjamini-Hochberg multiple com- for structurally unnamed biochemicals, which have been parison adjustment (FDR P value adjustment set at 0.05). identified by virtue of their recurrent nature (both chroma - tographic and mass spectral). A data normalization step was Abbreviations performed to correct variation resulting from instrument ART : Antiretroviral therapy; HEU: HIV‑ exposed uninfected; HUU: HIV‑unex ‑ inter-day tuning differences. Essentially, each compound posed uninfected; IMC: Infant meconium; IST: Infant stool; IOS: Infant oral swab; MST: Maternal stool; MVS: Maternal vaginal swab; MBM: Maternal breast was corrected in run-day blocks by registering the medians milk; PCoA: Principal coordinates analysis; PERMANOVA: Permutational multi‑ to equal one (1.00) and normalizing each data point propor- variate analysis of variance; ASV: Amplicon sequence variant; ANOVA: Analysis tionately (termed the “block correction”). of variance; FDR: False discovery rate; UPLC: Ultra‑performance liquid chroma‑ tography; SI: Shannon index; MaAsLin2: Multivariable association with linear models; WAZ: Weight‑for ‑age z‑score; TMP ‑SMX: Trimethoprim‑sulfameth‑ Statistical analysis oxazole; TRP: Tryptophan; KP: Kynurenine pathway; OPLS‑DA: Orthogonal partial least square discriminant analysis; PCA: Principal component analysis; Statistical analyses were performed using R (version 3.6.0). WHO: World Health Organization; MEBA: Multivariate empirical Bayes analysis; Demographic and clinical characteristics were compared UHPLC/MS/MS: Ultra‑high‑performance liquid chromatography/mass spec‑ between HEU and HUU children and their mothers using trometry/mass spectrometry; RP: Reverse‑phase; ESI: Electrospray ionization; MS: Mass spectrometry; QC: Quality control; HILIC: Hydrophilic interaction. Wilcoxon, Fisher’s exact and t tests. Longitudinal compar- isons of alpha diversity were performed using univariable Supplementary Information and multivariable linear regression. Pairwise compari- The online version contains supplementary material available at https:// doi. sons were performed with post hoc Tukey HSD test with org/ 10. 1186/ s40168‑ 022‑ 01230‑1. FDR P value adjustment set at level 0.05. Principal coor- dinates analysis (PCoA) using Bray-Curtis dissimilarity Additional file 1: Figure S1. Microbiota of 272 mother ‑infant pairs. was performed to assess the beta diversity. Permutational Figure S2. Shannon diversity of 272 mother‑infant pairs. Figure S3. multivariate analysis of variance (PERMANOVA) was PCoA of maternal microbiota based on HIV status. Figure S4. Shannon diversity of maternal microbiota based on HIV status. Figure S5. Maternal conducted to test whether the bacterial communities microbiota composition showed no significant differences based on sequenced have different centroids based on HIV-status HIV status. Figure S6. Number of breastfeeding and non‑breastfeeding (mothers) or HIV-exposure (infants). Significance of the infants throughout the 18‑month study period. Figure S7. PCoA of infant microbiomes based on HIV exposure, breastfeeding status, and time point. results was confirmed with a test of heterogeneity (ensure Figure S8. PCoA of infant microbiomes based on Bifidobacterium longum homogenous dispersion). In addition, multivariate asso- relative abundance, breastfeeding status, and time point. Figure S9. Shan‑ ciation with linear models (MaAsLin2) [112], an addi- non diversity of infant gut microbiota based on HIV exposure, breastfeed‑ ing status, and time point. Figure S10. Orthogonal partial least square tive general linear model with boosting that can capture discriminant analysis (OPLS_DA) and principal component analysis (PCA) the effects of a parameter of interest while deconfound - of breast milk. Figure S11. Orthogonal partial least square discriminant ing the effects of other metadata, was used to efficiently analysis (OPLS_DA) and principal component analysis (PCA) of breast milk at six weeks postpartum and 6 months postpartum. Figure S12. Top 20 determine multivariable association between clinical metabolites with highest Hotelling’s T value. Table S1. Sample collection metadata, 16S rRNA gene sequence data, and breast milk schedule. Table S2. Two‑ way repeated measures analysis of variance metabolomic data. MaAsLin2 analysis for the infants (ANOVA) of breast milk metabolites. was adjusted for delivery type, prematurity, timepoint, Gr ant‑Beurmann et al. Microbiome (2022) 10:61 Page 15 of 18 and uninfected, 2000‑18: a modelling study. Articles Lancet Glob Acknowledgements Health. 2020;8:67–75. The authors would like to thank the Genomics Resource Center at the 3. Mcgrath CJ, Nduati R, Richardson BA, Kristal AR, Mbori‑Ngacha D, Institute for Genome Sciences (University of Maryland School of Medicine) Farquhar C, et al. The prevalence of stunting is high in HIV‑1‑ exposed for the 16S rRNA amplicon sequencing, as well as gratefully acknowledge uninfected infants in Kenya 1‑3. J Nutr. 2012;142:757–63. the work of the Microbiome Service Lab and Jonathan Lim with running 4. Marinda E, Humphrey JH, Iliff PJ, Mutasa K, Nathoo KJ, Piwoz EG, et al. the sequencing data through their bioinformatics pipeline. 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Wei R, Msamanga GI, Spiegelman D, Hertzmark E, Baylin A, Manji K, et al. analysis of the metabolomics data. SG, OAM, CMF were responsible for the Association between low birth weight and infant mortality in children acquisition and/or analysis of microbiome data. SG, JJ, CMF, MC conducted born to human immunodeficiency virus 1‑infected mothers in Tanza‑ the statistical analyses. All authors participated in drafting and revising the nia. Pediatr Infect Dis J. 2004;23:530–5. manuscript and have read and approved the final manuscript. 8. Bender JM, Li F, Martelly S, Byrt E, Rouzier V, Leo M, Tobin N, Pannaraj PS, Adisetiyo H, Rollie A. Maternal HIV infection influences the microbiome Funding of HIV‑uninfected infants. Sci Transl Med. 2016;8:349ra100. The research reported in this publication was supported by the National 9. Machiavelli A, Duarte RTD, de Souza Pires MM, Zárate‑Bladés CR, Pinto Institute of Dental and Craniofacial Research of the National Institutes of AR. The impact of in utero HIV exposure on gut microbiota, inflamma‑ Health under Award Number R01DE025174. The content is solely the responsi‑ tion, and microbial translocation. Gut Microbes. 2019;10:599–614. bility of the authors and does not necessarily represent the official views of 10. González R, Mandomando I, Fumadó V, Sacoor C, Macete E, Alonso PL, the National Institutes of Health. Additional support was provided by the et al., editors. Breast milk and gut microbiota in African mothers and Dean’s Endowed Professorship to Claire M. Fraser, PhD (University of Maryland infants from an area of high HIV prevalence. PLoS One. 2013;8:e80299. School of Medicine). Olivia A. Martin is a TL1 post‑ doctoral fellow supported https:// doi. org/ 10. 1371/ journ al. pone. 00802 99. by 1TL1TR003100‑01, 1UL1TR003098‑01, and the University of Maryland, 11. Prendergast AJ, Chasekwa B, Rukobo S, Govha M, Mutasa K, Ntozini Baltimore, Institute for Clinical and Translational Research. R, et al. 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Microbiome – Springer Journals

Published: Apr 12, 2022

Keywords: HIV-exposed infants; Gut microbiota; Breast milk metabolome; Antiretroviral therapy; Breastfeeding; Bifidobacterium; Adverse growth outcome; Weight-for-age z-score; Acylcarnitine; Kynurenine

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Dynamics of the infant gut microbiota in the first 18 months of life: the impact of maternal HIV infection and breastfeeding

Dynamics of the infant gut microbiota in the first 18 months of life: the impact of maternal HIV infection and breastfeeding

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Dynamics of the infant gut microbiota in the first 18 months of life: the impact of maternal HIV infection and breastfeeding

Dynamics of the infant gut microbiota in the first 18 months of life: the impact of maternal HIV infection and breastfeeding

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