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). The vast majority (86.8%) of infants were exposed to maternal antibiotics, administered within 72 hours prior to birth. Twenty-two (32.4%) infants received postnatal antibiotics (classified as antibiotic-exposed) while 46 (67.6%) infants did not (antibiotic-naïve; Table 1). In the first week after birth, Ampicillin (18 infants, 26.5%), Nafcillin (4, 5.9%), Gentamicin (17, 25.0%), Tobramycin (2, 2.9%), and Vancomycin (2, 2.9%), were administered, while 48 infants (70.6%)) received no antibiotics. In the second to the third weeks - Ampicillin (1, 1.6%), Nafcillin (8, 12.9%), Gentamicin (1, 1.6%), Tobramycin (7, 11.3%), Vancomycin (2, 3.2%), and Amoxicillin (1, 1.6%) were administered, while 51 infants (82.3%) received no antibiotics (Supplemental Table 1). Antibiotic-naiive and antibiotic-exposed infants were similar in demographic characteristics except for modest difference in body weights which weas accounted for using generalized linear mixed modeling in subsequent analysis. Similarly, median specimen collection times were were not statistically signifacantly different, Table 1. Most (97.1%) of the infants were fed human milk during the study period. Two neonates developed NEC in the antibiotic-exposed group during the study period. Following shotgun sequencing, and after quality control (Supplemental Table 2 ), 375 samples were available for downstream analysis (Supplemental Table 3 - Genera, Supplemental Table 4 - Species, and Supplemental Table 5 - Pathways). A summary sample size by body sites, postnatal ages, gestational ages, antibiotic treatment groups and collection times, is provided in Supplemental Table 2.
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 in days
|
|
|
|
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
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 89 stool and 169 skin swabs (86 from the groin and 83 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 (Figure 1A). Similarly, comparison of samples at Week 1 and Week 3 postnatal ages by principal component analysis (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 (Figure 1B). As might be expected, the composition of groin skin microbiome was more similar to gut 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 gut microbiome in antibiotic-naïve infants. We present organism abundance data at the genus level to reduce complexity, given the larger number of species differentially abundant between Week 1 and Week 3 (species-level analysis revealed 236 species that were significantly different between Week 1 and Week 3 with FDR < 0.05; Supplemental Table 4). 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 (gestational age, maternal antibiotics, route of delivery and infant diet). Several other genera, Klebsiella, Veillonella, Serratia, Escherichia, were also significantly increased (Figure 2A and 2B, all p<0.05). Conversely, Staphylococcus demonstrated the greatest decrease in abundance from Week 1 to Week 3 (Figure 2C).
Impact of gestational and postnatal ages on developmental trajectory on gut 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 Figure 1A) whereas postnatal age had a significant impact on microbiome composition at all body sites among gestational age cohorts (Supplemental Figure 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 attained statistical significance as assessed by MRPP (Figure 3A). However, the trends for both developmental variables were in the same direction- in all cases in this PCA analysis (compare the orientation of directional arrows in Figures 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 (Figure 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 (Figure 3B). Overall, the data indicate that postnatal age had a greater impact on microbiome composition than gestational age, but there is a trend toward increased contribution of gestational age later in pregnancy. We also compared the contribution of gender to microbiome composition by performing unsupervised PCA. We found the only time point with a significant difference, based on gender, was the groin at Week 1 (p=0.002). The other body sites and time points were not significantly different with respect to gender (Supplemental Figure 2).
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. Stool samples showed a decrease in diversity during the first and third weeks and skin samples from the groin demonstrated decreased diversity by the third week post-antibiotic therapy (p<0.001, Figure 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 (Figure 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 (Figure 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 (Figure 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 in gut microbiota samples 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 (Figure 5A and Supplemental Figure 3). At week three, no genus was significantly higher in 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 in stool samples (Figures 5B and Supplemental Figure 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 (Figures 6A). In antibiotic-treated infants, E. coli and Klebsiella dominate at both time points (Figures 6B).
Effect of antibiotic exposure on maturation of metabolic functional capacity of gut 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 pathways content in antibiotic-naïve versus antibiotic-exposed. DNA reads were assigned to metabolic pathways using HUMAnN2 [21]. To assess overall metabolic pathway abundance, we used unsupervised PCA comparing samples based on the infant’s postnatal age and antibiotic exposure status (Figure 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. On PCA analysis, 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 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 (Figure 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 Figure 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 acids butyrate (PWY.5676. acetyl.CoA.fermentation.to.butanoate.II), and acetate (PWY.6588.pyruvate.fermentation.to.acetate) (Figure 8, and Supplemental Table 5). It is particularly notable that antibiotic exposure completely suppressed increased abundance of the butyrate and acetate synthesis pathways seen in antibiotic-naïve infants, suggesting that maturation of the gut microbiota from Week 1 to Week 3 is associated with increased capacity to produce metabolites beneficial to the developing neonatal intestinal epithelium (Figure 8). HUMAnN2 was unable to unambiguously identify organisms that contributed the most to short chain fatty acid synthesis, however Clostridium and Blautia species are major short chain fatty acid producers in the healthy gut microbiome [22, 23] and both were increased markedly in abundance in the gut of antibiotic-naïve infants from Week 1 to Week 3 (Figure 2A). Failure of this maturation in the gut microbiome of antibiotic-exposed infants (Figures 5B and Supplemental Figure 3) likely accounts for the marked defect in short chain fatty acid synthesis capacity in antibiotic exposed infants.