Study population and antibiotic exposure
Samples from the skin (axilla and groin) and stool were collected from 68 preterm infants, median (range) birthweight 1384 (648–1940) g and gestational age 30.5 (24.0–35.0) weeks (Table 1). Fifty-nine (86.8%) infants were exposed to maternal antibiotics, administered within 72 hours prior to birth. In addition, 22 (32.4%) infants received postnatal antibiotics (classified as antibiotic-exposed) while 46 (67.6%) infants did not (classified as antibiotic-naïve; Table 1). Infants in both groups were similar in demographic characteristics and sample collection times, except that antibiotic-exposed infants were modestly smaller than antibiotic-naïve infants, Table 1. This was accounted for using generalized linear mixed modeling in subsequent analysis. Most (97.1%) of the infants were fed human milk during the study period. Two neonates developed necrotizing enterocolitis (NEC) in the antibiotic-exposed group during the study period.
Table 1
Demographic characteristics
| Antibiotic-naïve group (n = 46) | Antibiotic-exposed group (n = 22) | p-values |
Birth weight in g, median (range) | 1478.0 (688–1940) | 1173.0 (648–1924) | 0.003 |
Gestational age in weeks, median (range) | 31.5 (28–35) | 28.5 (24–32) | < 0.001 |
Female gender, n (%) | 25 (54.3) | 12 (54.5) | 0.988 |
Multiple gestations, n (%) | 35 (76.1) | 17 (77.3) | 0.914 |
Race, n (%) | | | |
White | 23 (50.0) | 11 (50.0) | 1 |
Black or African American | 18 (39.1) | 10 (45.5) | 0.62 |
Asian | 2 (4.3) | 0 (0.0) | 0.321 |
Native Hawaiian or other Pacific Islander | 1 (2.2) | 0 (0.0) | 0.486 |
Unknown or not reported | 2 (4.3) | 1 (4.5) | 0.97 |
Hispanic or Latino, n (%) | 2 (4.3) | 1 (4.5) | 0.97 |
Cesarean section, n (%) | 42 (91.3) | 20 (90.9) | 0.957 |
Diet, n (%) | | | |
Mostly breast milk | 39 (84.8) | 19 (86.4) | 0.863 |
Any receipt of formula | 4 (8.7) | 1 (4.5) | 0.54 |
Any receipt of breast milk | 44 (95.7) | 22 (100.0) | 0.321 |
Perinatal maternal antibiotic exposure, n (%) | 39 (84.8) | 20 (90.9) | 0.486 |
Specimen Collection time | | | |
Week 1 | 7 (5–9) | 6.5 (5–9) | 0.907 |
Week 3 | 16 (15–21) | 17 (15–24) | 0.562 |
Maturation And Differentiation Of The Microbiome In Preterm Infants
To understand the ontogeny of the microbial community of the stool and skin in the preterm infant during the first three weeks of age, we focused on infants that did not receive postnatal antibiotics during this 3-week period. Microbial DNA from a total of 90 fecal and 180 skin swabs (90 from the groin and 90 from the axillae) from 46 antibiotic-naïve infants was sequenced and data from week 1 were compared with week 3. We found, as expected, that at all three body sites examined, the number and diversity of genera increased from week 1 to week 3 (Fig. 1A). Similarly, comparison of samples at Week 1 and Week 3 postnatal ages by PCA revealed maturation and differentiation at the three body sites. While at Week 1, samples from the three body sites were more closely clustered together, by Week 3, microbial composition had become more distinct across the three sites (Fig. 1B). As might be expected, the composition of groin skin microbiome was more similar to stool microbiome than was axillary skin microbiome, especially at Week 3. The composition at each body site at week 3 was distinct from composition at week 1 (multi-response permutation procedures (MRPP, p-values < 0.001 for Week 1 versus Week 3 stool, axilla, and groin microbial composition).
We next investigated which organisms made the largest contribution to microbiome maturation with a focus on fecal microbiome in antibiotic-naïve infants. Among the genera that significantly changed from Week 1 to Week 3, Clostridium demonstrated the most significance in abundance in week 3 after accounting for potential confounders. Several other genera, Klebsiella, Veillonella, Serratia, Escherichia, were also significantly increased (Fig. 2A and 2B, all p < 0.05). Conversely, Staphylococcus demonstrated the greatest decrease in abundance from Week 1 to Week 3 (Fig. 2C).
Impact of gestational and postnatal ages on developmental trajectory on stool microbiome composition
We examined the contribution of gestational age to microbiome composition at Weeks 1 and Weeks 3 in antibiotic-naïve infants. We found diversity and overall composition of the microbiome were not significantly different between infant born at 28–32 weeks compared with 33–36 weeks gestational age ranges (Supplemental Fig. 1A) whereas postnatal age had a significant impact on microbiome composition at all body sites among gestational age cohorts (Supplemental Fig. 1B).
We directly compared the contribution of gestational age versus postnatal age to microbiome composition by performing unsupervised PCA, then coloring samples based on gestational age in 2-week gestational age increments, as well as postnatal age at Weeks 1 and 3. Further, we calculated the mean Bray-Curtis distance of all pairwise comparisons between the two-week gestational age cohorts. Three trends are observed in the data, although none of the differences in microbial composition between any two-week gestational age cohorts were statistically significant as assessed by MRPP (Fig. 3A). However, the trend for both developmental variables was in the same direction- in all cases in this PCA analysis (compare the orientation of directional arrows in Figs. 3A and 3B). We then compared the impact of postnatal age on microbiome composition for each gestational age (GA) cohort in antibiotic-naïve infants. Microbiome composition demonstrated progression from Week 1 to Week 3 at all gestational ages (Fig. 3B). Postnatal age-dependent differences in microbiome composition were greatest at the earliest gestational ages (p = 0.017 for 28-to-30-week GA infants and p = 0.001 for 30-to-32-week infants) and declined with advancing gestational age (Fig. 3B). Overall, the data indicate that two postnatal weeks time period had a greater impact on microbiome composition than two gestational weeks, but there is a trend toward increased contribution of gestational age later in pregnancy.
Effect of perinatal maternal antibiotic exposure on neonatal microbiome structure
In our cohort, although most infants did not receive antibiotics postnatally, 86.8% of mothers received antimicrobials prior to delivery. We evaluated the effects of maternal antibiotics on the developing skin and gut microbiome and found no significant difference in Shannon Diversity over the sampling period in gut microbiome in infants who had been exposed to maternal antibiotics, administered within 72 hours prior to birth compared to those who were not. Maternal antibiotics have a non-significant trend toward decrease diversity at all body sites at Week 1, but this had resolved by Week 3 (Supplemental Fig. 2A). Similarly, PCA failed to reveal significant difference in the microbial composition and maturation of the microbiome at any of the three body sites (Supplemental Fig. 2B; only stool and groin samples are shown for clarity). However, we found using ZINB-GLMM, that prenatal antibiotic exposure was associated with alterations in abundance of several genera after accounting for other variables, including gestational age, breast milk receipt, and delivery mode both at week 1 and week 3 (Supplemental Fig. 2C).
Effect Of Antibiotic Treatment On Microbiome Diversification And Maturation
We next sought to understand how postnatal antibiotic exposure impacted composition and maturation of the preterm infant gut and skin microbiota. Unlike prior investigations of the preterm infant microbiome, the majority of infants in our study received no antibiotics up to 3 weeks of age. Among those that received postnatal antibiotics, the duration and intensity were generally short. Indeed, among the 22 infants that received antibiotics, 16 infants (72.7%) received < 48 hours of ampicillin and gentamicin, and predominantly during the first 3 days of age. We first compared microbiome diversity across body sites at Week 1 and Week 3 between antibiotic-exposed and antibiotic-naïve groups. Fecal samples showed a decrease in diversity during the first and third week and skin samples from the groin demonstrated decreased diversity by the third week post-antibiotic therapy (p < 0.001, Fig. 4A). There were no significant differences in diversity of microbiota from the axillae at Week 1 and Week 3 between antibiotic-exposed and antibiotic-naïve groups. We next examined overall microbial composition using PCA. There was decreased differentiation of the gut microbial composition in antibiotic-exposed infants compared to antibiotic-naïve infants (Fig. 4B). Overlay of the PCA plots from Week 1 and Week 3 demonstrates that antibiotic treatment tends to shift microbial composition in the opposite direction relative to changes seen with postnatal age, indicating that antibiotics therapy is associated with blunting of the maturation of the microbiome at all three body sites (Fig. 4C). In addition, microbiome maturation across body sites measured by Bray–Curtis distance shows significant decrease in differentiation (p < 0.001) between antibiotic-exposed and antibiotic-naïve groups from week 1 to week 3 (Fig. 4D).
To identify organisms most impacted by antibiotic exposure, we performed ZINB-GLMM with FDR correction followed by calculation of effect size using SLDA to identify genera that differed between the microbial composition of antibiotic-exposed versus antibiotic-naïve infants at Weeks 1 and 3, after accounting for gestational age, maternal antibiotics, breast milk receipt, and delivery mode. We found that Sphingomonas, Acidovorax and Candida were significantly enriched in the antibiotic-exposed group, whereas several genera, including Blautia, Streptococcus, Enterococcus and Staphylococcus were significantly more abundant in the antibiotic-naïve infants in the first week after birth (Fig. 5A and Supplemental Fig. 3). At week three, there were no genera found at significantly higher abundance in antibiotic-exposed infants, while several genera including Clostridium, Clostridioides, Blautia, as well as Streptococcus, and Staphylococcus were significantly increased in the antibiotic-naïve infants at three weeks after birth (Figs. 5B and Supplemental Fig. 3). Antibiotic exposure resulted in domination of the gut microbiome by a small number of genera, as indicated by the Berger-Parker Dominance index both at week 1 and week 3 (Figs. 6A). In antibiotic-treated infants, E. coli and Klebsiella dominate at both time points (Figs. 6B).
Effect of antibiotic exposure on maturation of metabolic functional capacity of stool microbiome
Microbes are believed to influence human health, in part, through their ability to produce metabolites with either beneficial or harmful effects on the host. Given the primacy of metabolic activity in the preterm infant gut microbiota and potential direct impacts in health outcome, we characterized the metabolic gene content in antibiotic-naïve versus antibiotic-exposed. DNA reads were assigned to metabolic genes using HUMAnN2 [7]. To assess overall metabolic pathway abundance, we used unsupervised principal component analysis (PCA) comparing samples based on the infant’s postnatal age and antibiotic exposure status (Fig. 7A). There was a significant change in the distribution of metabolic pathways represented in the preterm infant gut between Week 1 and Week 3 in antibiotic-naïve infants (p < 0.001), indicating the metabolic capacity demonstrated significant functional maturation in these infants. However, the change from Week 1 to Week 3 in antibiotic-exposed infants was not significant (p = 0.064), indicating that antibiotics impaired maturation of metabolic functional capacity. PCA analysis demonstrated that antibiotic exposure was associated with a shift to a more immature metabolic pathway abundance at Week 3 compared to antibiotic-naïve infants. These two features of the PCA plot reveal that antibiotic exposure is associated with stunting of functional maturation of the preterm infant microbiome. To specifically address the role of antibiotic exposure and account for differences in gestational age, maternal antibiotic exposure between groups, we employed generalized linear mixed modeling (GLMM) with fixed and random effects. We found that genes encoding a total of 20 metabolic pathways that were significantly different from Week 1 to Week 3 in the stools of antibiotic-naïve infants. In contrast, 34 metabolic pathways were significantly different in infants that received antibiotics (Fig. 7B). Notably, there was very little overlap in the metabolic pathways enriched from Week 1 to Week 3 in antibiotic-naïve versus antibiotic-exposed infants (Supplemental Fig. 4) indicating that antibiotic exposure is associated with profoundly different trajectories in the development of metabolic functional pathways in the preterm infant gut. Among metabolic pathway genes that were significantly increased in antibiotic-naïve infants were pathways involved in synthesis of the short chain fatty acid butyrate (PWY.5676. acetyl.CoA.fermentation.to.butanoate.II), such as butyrate and acetate pathway, Fig. 8. It is particularly notable that antibiotic exposure completely suppressed increased abundance of the butyrate and acetate synthesis pathway seen in antibiotic-naïve infants, suggesting that maturation of the fecal microbiota from Week 1 to Week 3 is associated with increased capacity to produce metabolites beneficial to the developing neonatal intestinal epithelium (Fig. 8).