1. Introduction
Gut microbiota plays a crucial role in infant health and development. During the first six months of life, the gut microbiota undergoes highly dynamic changes, reaching a stable, adult-like state by 2–3 years of age [
1,
2]. Disturbances to early life gut microbiota have been associated with increased risks of chronic conditions, including obesity, metabolic syndrome, diabetes, and immune system disorders [
3,
4,
5]. As such, promoting healthy gut microbiota development in infancy may be crucial for lifelong well-being. Researchers have increasingly focused on how antibiotics and probiotics impact the infant’s gut microbiota [
6,
7]. Understanding these interventions may provide valuable insights for mitigating disease risks in childhood and adulthood.
Antibiotics are commonly prescribed to infants to treat bacterial infections, beginning at the time of delivery. Specifically, intrapartum antibiotic prophylaxis (IAP) is a clinical measure used in more than 30% of deliveries to prevent group B Streptococcus (GBS) infection that may be administered before or during a cesarean section (CS) to prevent infection [
8,
9]. However, researchers have revealed adverse effects of IAP, including delayed microbial maturation and altered microbiota establishment [
10,
11]. Furthermore, children are highly prescribed antibiotics in China (43.5%) at a rate higher than those reported in many countries, such as France (26.1%) and Australia (23%) [
12]. Antibiotic exposure during early life has been linked to disrupted microbial colonization, decreased microbial diversity and stability, increased risk of gut dysbiosis and chronic diseases, and elevated levels of antibiotic resistance genes (ARGs) [
13,
14,
15].
Studies have shown that probiotics have potential significance in modulating and clinically counteracting antibiotics’ adverse effects on the gut microbiota [
16,
17]. However, probiotic co-prescription rates remain low, particularly in the Asia–Pacific region [
18]. Controlled trials in infants have shown that probiotic interventions increase the relative abundance of
Bifidobacterium infantis and
Lactobacillus, restore microbial diversity, decrease the relative abundance of pathogenic commensal bacteria, and reduce antibiotic resistance in gut microbiota, indicating better restoration of the child [
17,
19,
20]. Yet, the efficacy of probiotics in treating microbiota dysbiosis in infants varies in clinical practice. Specific probiotic strains have demonstrated no significant benefit over the placebo in eradicating antibiotic-resistant colonization, and probiotic genome analyses have identified potential risks of spreading antibiotic resistance [
21,
22].
Previous studies have often examined antibiotics and probiotics separately, using cross-sectional or retrospective designs with limited samples and durations. This highlights the need for research on their combined effects on gut microbiota and antibiotic resistance in infants under six months, particularly in rural areas with high rates of antibiotic misuse. This study aimed to investigate the distinct impacts of antibiotic and probiotic administration on the composition and structure of gut microbiota and the prevalence of six commonly used ARGs in infants within the initial six months of life based on a well-characterized birth cohort.
3. Discussion
In this study, we characterized the composition of gut microbiota and the presence of ARGs in infants at three time points in the first six months of life. Due to loss to follow-up at two and six months of age, lost respondents were distributed across antibiotics and probiotics use groups; they were not statistically different from follow-up respondents in terms of mother and infant characteristics and, thus, would have no impact on the results. By exploring correlations between gut microbial communities, ARGs, and exposure to antibiotics and probiotics, we sought to elucidate potential interactions among these factors. Our findings revealed that the use of antibiotics and probiotics can significantly impact the core genera, and while antibiotic exposure was associated with altered ARG abundances, probiotic intake did not exhibit such effects. Additionally, we were able to identify putative microbial hosts for specific ARGs. These results enhance our understanding of the complex interplay between clinical interventions, the develo** gut microbiota, and the emergence of antimicrobial resistance in early life, providing insights to guide evidence-based strategies for optimizing infant gut health and mitigating resistance risks.
The postnatal colonization and assembly of the infant gut microbiota is a highly dynamic process, and antibiotic exposure during this critical window can disrupt gut homeostasis, leading to the depletion of keystone taxa, diminished taxonomic diversity, altered metabolic functions, and the potential proliferation of pathogenic organisms [
23]. In the present study, the predominant phyla in infants were found to be Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria, which mirrored compositions reported previously [
24]. Interestingly, further stratification by delivery mode revealed an upregulation of the genus
Lactococcus associated with the CON group under the VD subgroup. Notably, a previous study reported a
Lactococcus strain isolated from the maternal vagina that exhibited probiotic properties [
25], which implies that antibiotic exposure during delivery may disrupt the vertical transmission of beneficial taxa like
Lactococcus from the maternal to infant gut microbiota. We did not observe notable disparities in alpha and beta diversity in the first three days of life or at the two-month time point. This may be attributable to the uniform colonization and development characteristics of this early period. Alternatively, the effects of antibiotics or probiotics may not yet be manifested in such young infants, as prior research has demonstrated limited impacts of early-life probiotic exposure on gut microbiota diversity [
26,
27]. The results of the present study align with these previous findings.
Core microbiome genera are widely distributed and abundant across samples. Consistent with prior research [
28,
29,
30], we found that exposure to IAP reduced the abundance of the core genus
Bifidobacterium in infants within three days of age. Conversely, in healthy full-term infants without IAP exposure,
Bifidobacterium dominated the gut. Furthermore, IAP has been shown to decrease
Bifidobacterium over time while increasing opportunistic pathogens like
Clostridium difficile [
10,
31]. This
Bifidobacterium depletion may promote gut dysbiosis, including an elevated pH and proliferation of spore-forming bacteria [
32]. Importantly, we observed a higher
Enterococcus_faecium abundance in the probiotic-supplemented group at two months. As
Enterococcus is commonly used clinically as a probiotic [
33], this likely reflects the exogenous supplementation. By four to six months, as solid foods are introduced, genera like
Helicobacter and
Clostridium are typically established [
34,
35]. Interestingly, probiotic intake may competitively inhibit or modulate the gut environment to reduce these genera. Further mechanistic research is warranted to fully elucidate probiotic impacts on the infant gut microbiota.
The present study found a high prevalence of antibiotic resistance genes (ARGs) in the infant gut microbiota, which is consistent with previous longitudinal research detecting ARGs for aminoglycosides, beta-lactams, macrolides, and tetracyclines across the first year of life [
36]. These ARG variations may stem from resistant bacterial strains present harboring specific ARGs that can spread to other strains. Additionally, infant exposure to environmental antibiotic residues may enhance the competitive advantage of resistant microbes. Notably, we did not find evidence that probiotic supplementation reduced ARG abundance, aligning with prior reports [
37]. This may be explained by the variable antibacterial properties and intrinsic resistance profiles of different probiotic strains [
38,
39,
40]. Thus, when selecting probiotics, it is critical to not only identify the species but also characterize their resistance determinants.
Previous work has postulated that ARGs could potentially find host organisms within microbial communities based on significant correlations between ARG genes and similarities of abundances, which were observed across various samples (
p < 0.01;
r > 0.6) [
41]. Consistent with this, we observed associations between ARGs and the gut microbiota in the present study, suggesting that the microbiota harbors antibiotic resistance. In recent years, correlation analysis has been widely conducted to infer ARG hosts in fecal samples [
42]. To build on these observations, future research should integrate sequencing technologies with functional metagenomics or genome assembly approaches to further validate ARG–microbe linkages.
In summary, the present study provides preliminary evidence linking antibiotics, probiotics, gut microbiota, and ARGs. This finding may be particularly relevant in regions with high antibiotic usage, highlighting the need for the appropriate regulation of these interventions. The longitudinal design enabled the observation and analysis of the microbial composition and antibiotic resistance within the first six months of infant life. The comprehensive dataset, including high-throughput sequencing, usage records, and resistance gene detection, offered insights into the effects of antibiotics and probiotics on the gut microbiota. However, there were some limitations to our study. First, the infant numbers in the antibiotics and probiotics groups were relatively small, and the group that used both antibiotics and probiotics was not shown due to the limited number in the follow-up samples. It is necessary to increase the number of findings to provide insights for future studies with larger sample sizes to elucidate the effects of combining antibiotics and probiotics for intervention. Second, qPCR methods have limitations compared to metagenomic sequencing for characterizing the resistome and microbiota, but this targeted and cost-effective technique provides insights into our future utilization of metagenomic sequencing to determine antibiotic resistance profiles. Third, though infants within six months have a relatively simple environment, potential confounding factors in the study may not be adequately controlled and can affect the interpretation of results, so it is difficult to establish causality. Furthermore, this study considered the cross-sectional exposure of antibiotics and probiotics without residual effects alongside the consideration of earlier exposures, which need to be analyzed further in a larger sample. Lastly, according to the WHO classification, combining Access (penicillins and cefazolin) and Watch (cefotiam) antibiotics in the analysis led to different attributable risks due to neglecting the prevalence of usage. Specifying the AWaRe category and analyzing it separately is crucial to highlight their appropriate use and public health significance. Therefore, further research is needed to better elucidate the mechanisms by which antibiotics and probiotics shape the develo** gut microbiota and antibiotic resistance, emphasizing their public health implications.