1. Overview of Illumina sequencing
In general, 55 samples (25 breast milk samples and 30 fecal samples) were sequenced using Miseq platform and 1,482,182 optimized sequences were acquired after quality-filter. Overall, 161 OUTs were classified for the following analysis on the basis of 97% similarity.
2. Microbiota in breast milk after perinatal antibiotic treatments
The disturbance to the microbiota in breast milk after perinatal antibiotic treatments was assessed based on the α-diversity indices. As illustrated in Fig. 1A-C, no significant difference in microbial community richness or diversity was observed in the breast milk samples from CXM treated or CXM + CFX treated mothers comparing with the control group. According to the unweighted-UniFrac dissimilarity based PCoA scores (Fig. 1D), there was no significant difference in the microbial community composition between the control group and CXM treated group (p > 0.05) according to the Adonis analysis.
The microbiome composition in breast milk at the phylum and genus levels across different groups are depicted in Fig. 2A, B. The bacterial community composition in breast milk was relatively simple, regardless of antibiotic treatments. Firmicutes were the most prevalent bacteria, followed by Actinobacteria. As to genus level, the most abundant bacterial genera were in order as follows: Streptococcus, Staphylococcus, and Rothia. The most vulnerable bacteria were identified by analyzing the differences in bacterial communities between groups. Generally, no dominating bacteria at the phylum or genus levels was identified in breast milk samples from CXM or CXM + CFX treated groups when compared to the control group using Kruskal-Wallis rank-sum test. LEfSe analysis had reflected the significantly affected bacteria in breast milk (Fig. 2C, D). According to the LDA score at the genus level, the abundance of Pseudomonas was significant higher in breast milk samples from CFX-treated group (p < 0.05, LDA > 2), whereas Clostridiaceae-sensu-stricto was the most abundant bacteria in breast milk from CFX + CXM-treated group (p < 0.05, LDA > 2). Additionally, 25 significantly disturbed bacteria OTUs were identified in the microbiota of breast milk (Fig. 2E). In summary, CXM treatment upregulated 18 OTUs and downregulated 3 OTUs in breast milk microbiota comparing with that of the control group. In breast milk from CFX + CXM-treated group, 8 OTUs increased while 5 OTUs decreased comparing with control group.
3. Gut microbiota in neonates
Antibiotic treatments to mothers during the perinatal period seemed to induce more profound effects on the gut microbiota in neonates. Shannon index indicated no significant difference in the microbial community diversity in the fecal samples from control group and CXM-treated group, but the microbial community diversity in CXM + CFX treated group was significantly lower than that in control group and CXM-treated group. However, no significant difference was observed about the community richness of the gut microbiota in neonates from different groups (Fig. 3A-C). As shown in Fig. 3D, PCoA and Adonis analyses also revealed a huge discrepancy existed in the gut microbiota composition between control group and CXM + CFX-treated group (p < 0.001).
Meanwhile, the dominant bacterial community in fecal samples were more abundant than that in breast milk samples (Fig. 4A, B). The most abundant bacteria phyla in the fecal samples from control group followed the order of Bacteroidota, Proteobacteria, and Firmicutes. In antibiotic treatments groups (both CXM and CXM + CFX treatments), the most dominant bacteria phylum was Firmicutes followed by Proteobacteria. At the genus level, the most abundant bacteria genera in fecal samples from control group were Bacteroides, Escherichia-Shigella, and Clostridium-sensu-stricto. The dominant bacteria genera in CXM-treated group were Streptococcus, Clostridium-sensu-stricto, Escherichia-Shigella, Enterobacteriaceae-unclassified, and Clostridioides. In CXM + CFX-treated group, the dominant bacteria genera were slightly different, which were Streptococcus, Enterobacteriaceae-unclassified, and Enterococcus. Kruskal-Wallis rank-sum test reflected that CXM treatment enhanced the abundance of Firmicutes while decreased Bacteroidetes abundance at the phylum level. As for genus level, CXM treatment reduced the abundance of Bacteroides, Escherichia-Shigella but increased the abundance of Clostridioides, Enterobacteriaceae-unclassified, Lactobacillales-unclassified, Streptococcus, Veillonella (Fig. 4C, D). The significance in bacterial community differences of CXM + CFX-treated group was subject to the limited clinical samples. As the Fig. 4E, F shows, further LEfSe analysis revealed that Bacteroides, Escherichia-Shigella, and Lactobacillus were the most different bacteria genera in fecal samples from control group (p < 0.05, LDA > 2). In CXM-treated group, the most abundant bacteria genera were Clostridioides, Faecalibacterium, Lactobacillales-unclassified, and Veillonella (p < 0.05, LDA > 2). As to CXM + CFX-treated group, the most susceptible bacteria genera were Enterobacteriaceae-unclassified and Streptococcus (p < 0.05, LDA > 2). Besides, the significantly disturbed bacteria OTUs by antibiotic treatments in fecal samples were up to 31 (Fig. 4G). Among these, OTU91 (Faecalibacterium), OTU19 (Pseudomonas), OTU17 (Veillonella), OTU4 (Clostridiaceae-sensu-stricto), OTU1 (Streptococcus), OTU76 (Pelomonas), and OTU11 (Enterobacteriaceae-unclassified) exhibited the consistent phenotype in breast milk and fecal samples.
4. Gut microbiota alternations in infants
The gut microbiome composition in infants showed a substantial recuperative trend in the follow-up visits. The microbial community richness and diversity of gut microbiota in the fecal samples of CXM-FV group were significantly higher than that of CXM-treated group (Fig. 5A-C). As illustrated in Fig. 5D, PCoA analysis in conjunction with Adonis analysis revealed a significant change in the microbial community composition between CXM-FV group and CXM-treated group (p < 0.001).
The dominant bacterial community in fecal samples of CXM-FV group was also prone to revert to the initial states. The most abundant bacteria phyla in the fecal samples from CXM-treated group were Firmicutes and Proteobacteria; while the dominant bacteria phyla in CXM-FV group followed the order of Actinobacteria, Firmicutes, and Proteobacteria (Fig. 6A). At the genus level, Escherichia-Shigella, Streptococcus, Clostridiaceae-sensu-stricto, Enterobacteriaceae-unclassified, Clostridioides were the most abundant bacterial genera in CXM-treated group, whereas Escherichia-Shigella and Bifidobacterium were the most abundant bacterial genera in CXM-FV group (Fig. 6B). The Wilcoxon rank-sum test revealed that Actinobacteria were considerably more abundant in CXM-FV group at the phylum level. At the genus level, CXM-FV group had higher abundance of Bifidobacterium and another six bacteria genera, but CXM-treated group had lower abundance of Clostridioides, Pseudomonas, and Firmicutes-unclassified (Fig. 6C, D). As shown in Fig. 6E, F, LEfSe analysis further confirmed that Clostridioides, Comamonas, Parabacteroides, Pseudomonas, and Firmicutes-unclassified were the most distinct bacteria genera in CXM-treated group (p < 0.05, LDA > 2), whereas Bifidobacterium and the remaining 18 bacterial genera were more abundant in CXM-FV group (p < 0.05, LDA > 2). In addition, 24 significantly altered bacteria OTUs were identified between CXM-treated group and CXM-FV group with only OTU19 (Pseudomonas) and OTU6 (Clostridioides) exhibiting lesser abundance in CXM-FV group (Fig. 6G).
5. ARGs abundance of gut microbiota in neonates
The ARGs abundance in neonatal fecal samples was examined to evaluate the antibiotic resistance in gut microbiota. The results of HT-qPCR demonstrated that antibiotic treatments to mothers during the perinatal period made a difference to the antibiotic resistance in neonatal gut microbiota (Fig. 7A). The relative abundances of ARGs were substantially higher in fecal samples from the control group than those from CXM-treated group. Over 50% of ARGs demonstrated declining trend in CXM-treated group (particularly blaOXA10, blaSHV, cfxA, tnpA-04, and tnti1), while most ARGs were considerably down-regulated in CXM + CFX-treated group.
6. ARGs transfer in gut microbiota of infants
The ARGs transfer in gut microbiota of infants were investigated by comparing the obtained results with the fecal samples from the follow-up visits. As illustrated in Fig. 7B, the relative ARGs abundance of infants’ gut microbiota presented a significant increasing trend (blaGES, blaTEM, tnpA-04, and tnti1) after a 6-month recovery period.