Breastfeeding and early Bifidobacterium-driven microbial colonization shape the infant gut resistome

Abstract

The assembly of the gut resistome in early life is key to infant health. Specific perinatal factors such as cesarean section (C-section), antibiotic exposure and lack of breastfeeding practices are detrimental to proper microbial development and increase the antimicrobial resistance genes (ARGs). Using 265 gut longitudinal metagenomes from 66 mother-infant pairs, we investigated how perinatal factors influence the acquisition and dynamics of ARGs during the first year of life. Our findings reveal that Bifidobacterium plays a crucial role in modulating the infant resistome, with its high relative abundance being associated with a lower ARG load. Exclusive breastfeeding during the first month of life accelerates the reduction of ARGs and ensures a lower resistome burden at six months. Moreover, early breastfeeding cessation correlates with a higher ARG load, underscoring its long-term influence on microbial resilience. Importantly, we identify exclusive breastfeeding as a key strategy to mitigate the impact of C-section delivery on the infant gut resistome, counteracting the early-life antibiotic exposure associated with this procedure and the resulting resistance acquisition. By promoting a microbiome enriched in Bifidobacterium, breastfeeding may help suppress ARG-carrying taxa, reducing the risk of resistance dissemination. Our findings underscore the importance of breastfeeding as a natural intervention to shape the infant microbiome and resistome. Supporting breastfeeding through public health policies could help limit the spread of antimicrobial resistance in early life.

Fig. 1. Mother-infant’s gut resistome during the first year of life.

A Abundance of assembled ARGs in mother-infant pairs at 1 month postpartum (N = 39 complete dyads), classified as shared, unique in mothers and unique in infants, and grouped by antibiotic class. B Antibiotic resistance load in the gut of infants during the first year of life and their mother’s, expressed as total CPM of ARGs per infant in each timepoint. ARG load was the summed abundance of all ARGs in a given sample. Significance values for ARG load were calculated using the pairwise rank-sum Wilcoxon test (Mann-Whitney U), two-sided. C Alpha diversity of the mother-infant gut resistomes based on a two-sided Kruskal-Wallis test on the Shannon index. D Composition of the infant resistome through time, coloured by the infant’s age. Two-sided PERMANOVA on Jaccard distances was applied to assess differences between timepoints (p < 0.05 for all comparisons). E Composition of the resistome at the antibiotic class level of mother-infant pairs. ARGs have been grouped according to the antibiotic(s) they confer resistance. All samples represent independent biological replicates, derived from individual participants. Sample sizes per group are as follows: day 7 infants (N = 58), 1-month-old infants (N = 58), 6-month-old infants (N = 56), 12-month-old infants (N = 55), and mothers (N = 39). Box plots show the median (centre line), the first and third quartiles (bounds of the box). Whiskers are chosen to show the 1.5 of the IQR. Asterisks indicate statistical significance: *p  <  0.05; **p  <  0.01; ***p  <  0.001; ****p  <  0.0001. FPA: Folate Pathway Antagonist. Source data are provided as a Source Data file.

Fig. 2. Infant’s gut microbiome during the first year of life and bacterial hosts of resistances.

A Alpha diversity of the mother-infant gut resistomes based on the Kruskal-Wallis test on the Shannon index. Box plots show the median (centre line), the first and third quartiles (bounds of the box). Whiskers are chosen to show the 1.5 of the IQR. All samples represent independent biological replicates, derived from individual participants. Sample sizes per group are as follows: day 7 infants (N = 58), 1-month-old infants (N = 58), 6-month-old infants (N = 56), 12-month-old infants (N = 55), and mothers (N = 39). Asterisks indicate statistical significance: *p  <  0.05; **p  <  0.01; ***p  <  0.001; ****p  <  0.0001. B Microbial composition at the species level of infants gut over the first year of life. Samples are coloured according to infants’ age. Two-sided PERMANOVA on Bray-Curtis distances was applied to assess differences between timepoints. p = 0.05 for 7 days compared to older timepoints. C Microbial hosts of resistance during the first year of life. ARG-carrying contigs were detected within the bins. D Antibiotic resistance and their microbial hosts during the first year of life. E Relative abundance (%) of genus carrying ARGs and number of copies of these genes associated with a plasmid. FPA: Folate Pathway Antagonist; MLSB: Macrolide-Lincosamide-Streptogramin B. Source data are provided as a Source Data file.

Fig. 3. Bifidobacterium abundance drives microbial composition.

A Genus relative abundance over time, depending on the Bifidobacterium abundance-based clusters. Two distinct clusters (optimal number of clusters k  =  2) were identified via k-means clustering. Mann-Whitney U test revealed that statistically significant differences in relative abundance were found in the Bifidobacterium genus, in all timepoints, so clusters were named as High- and Low-Bifidobacterium. Upper boxplots represent alpha diversity of the mother-infant gut resistomes based on a two-sided Kruskal-Wallis test on the Shannon index. Box plots show the median (centre line), the first and third quartiles (bounds of the box). Whiskers are chosen to show the 1.5 of the IQR. Sample sizes per group and timepoint were as follows: day 7: High-Bifidobacterium N  =  23, Low-Bifidobacterium N  =  35; 1 month: High N  =  42, Low N  =  16; 6 months: High N  =  31, Low N  =  25; 12 months: High N  =  19, Low N  =  36. Asterisks denote statistical significance: *p  <  0.05; **p  <  0.01; ***p  <  0.001; ****p  <  0.0001. B Stability of the hierarchical clustering based on Bifidobacterium spp. abundance over the first year of life. C Relative abundance of Bifidobacterium species in each cluster during the first year of life. Only species with statistically different abundances at any time point are shown (Mann-Whitney U test, *p  <  0.05). Source data are provided as a Source Data file.

Fig. 4. Bifidobacterium abundance drives resistance load and composition.

A Antibiotic resistance load in the gut of infants during the first year of life, depending on their Bifidobacterium abundance (cluster High or Low), expressed as total CPM of ARGs per infant in each timepoint. ARG load was the summed abundance of all ARGs in a given sample. Significance values for ARG load were calculated using the pairwise rank-sum Wilcoxon test (Mann-Whitney U), two-sided. Box plots show the median (centre line), the first and third quartiles (bounds of the box). Whiskers are chosen to show the 1.5 of the IQR. All samples represent independent biological replicates, derived from individual participants. Sample sizes per group and timepoint were as follows: day 7: High-Bifidobacterium N  =  23, Low-Bifidobacterium N  =  35; 1 month: High N  =  42, Low N  =  16; 6 months: High N  =  31, Low N  =  25; 12 months: High N  =  19, Low N  =  36. Asterisks denote statistical significance: *p  <  0.05; **p  <  0.01; ***p  <  0.001; ****p  <  0.0001. B Antibiotic resistance profiles in each group. ARGs have been grouped according to the resistance they confer. C Number of copies of ARGs associated with a plasmid in High- and Low-Bifidobacterium grouped infants. FPA: Folate Pathway Antagonist; MLSB: Macrolide-Lincosamide-Streptogramin B. Source data are provided as a Source Data file.

Fig. 5. The influence of environmental factors on shaping infant’s resistome during first year of life.

A Environmental factors driving infant’s resistome, ranked by effect size (‘envfit’ R2, false discovery rate (FDR)-corrected two-sided test, p < 0.05). B Antibiotic resistance load in the gut of infants of 7 days of age, depending on their mode of delivery (N = 40 vaginal, N = 26 C-section), expressed as total CPM of ARGs per infant in each timepoint. C Principal Coordinate Analysis (PCOA) of the composition of the resistome depending on the mode of delivery (vaginal or C-section) during the first 7 days and 1 month of life, based on Jaccard distances. Two-sided PERMANOVA on Jaccard distances was applied to assess differences between groups. D Antibiotic resistance load in the gut of infants of 1 month of age, depending on their diet (N = 40 breastfeeding, N = 4 formula feeding, N = 14 mixed), at each timepoint during the first year of life, expressed as total CPM of ARGs per infant in each timepoint. E Antibiotic resistance load in the gut of infants during the first month of life, depending on exclusive breastfeeding (N = 40 yes and N = 18 no), at each timepoint during the first year of life, expressed as total CPM of ARGs per infant in each timepoint. F Tendency of antibiotic resistance during the first year of life according to breastfeeding during the first month of life. The trajectory of the total load of ARGs was studied considering if the infant received exclusive breastfeeding during the first month of life. G ARGs differentially abundant due to breastfeeding in 6-month-old infants compared to non-breastfed infants, calculated using DESeq2 with the p-values attained by the Wald test corrected for multiple testing using the Benjamini-Hochberg method. H Antibiotic resistance load in the gut of infants during the first year of life, depending on the mode of delivery and if they were exclusively breastfed, at each timepoint during the first year of life, expressed as total CPM of ARGs per infant in each timepoint. I Multivariable association between factors and resistome composition using MaAsLin. Antibiotic resistance load was the summed abundance of all ARGs in a given sample. Box plots show the median (centre line), the first and third quartiles (bounds of the box). Whiskers are chosen to show the 1.5 of the IQR. Significance values for ARG load were calculated using the pairwise rank-sum Wilcoxon test (Mann-Whitney U), two-sided. Asterisks denote statistical significance: *p  <  0.05; **p  <  0.01; ***p  <  0.001; ****p  <  0.0001. kmeans_bifis: Bifidobacterium abundance-based clustering; AB_BB: infant’s antibiotic consumption; BF: breastfeeding; nBF_C: not exclusively breastfed infants born by C-section; nBF_V: not exclusively breastfed infants born vaginally; BF_C: exclusively breastfed infants born by C-section; BF_V: exclusively breastfed infants born vaginally. Source data are provided as a Source Data file.