People have recently paid increased attention to the colonization patterns of the gut microbiota in very premature infants. Previous studies have shown that establishing a normal gut microbiota is crucial for the health of very premature infants [20; 21], and dysbiosis of the gut microbiota is involved in NEC and LOS development [22; 23]. However, our results showed that B. animalis Bb-12 supplementation initiated early in life could modulate the colonization and development of the gut microbiota, which was mainly reflected in the significantly increased abundance of Actinomycetes and Bifidobacterium species. This was consistent with the results of Rahkola et al. [24], who showed that the relative abundance of B. animalis and Lactobacillus increased in the probiotic group 16 days after birth (P < 0.05). Therefore, early probiotic intervention can enrich the gut microbiota of very preterm infants, increase the number of Bifidobacterium and other probiotics, prolong the colonization time, and change the colonization pattern of the gut microbiota in very preterm infants. Compared with the control group, the results of this study showed that although Bifidobacteria in the observation group were significantly increased, the abundance of Streptococcus, Staphylococcus, Enterococcus, Escherichia, Klebsiella, and other pathogenic bacteria was not significantly reduced. Beck et al.[25] showed that probiotics drive microbiome transition into different preterm gut community types and inhibit the growth of pathogenic bacteria. Marißen et al.[26] found that probiotics could reduce the colonization of multidrug-resistant or gram-negative bacteria with high transmission potential, thus improving the short-term clinical outcomes of premature infants. No difference in pathogenic bacteria was found in our study, which was attributed to the small sample size and short follow-up time.
In this study, the Alpha diversity of the gut microbiota in the two groups was analyzed on days 14 and 28. The results showed that the Chao index of the gut microbiota in very preterm infants with probiotic supplementation increased on day 28 after birth. However, the Shannon index was not statistically significant. Further analysis showed that the weighted UniFrac beta diversity value in the observation group significantly increased. This means that the early intervention of probiotics increased the number of B. animalis and improved the richness and diversity of the gut microbiota, resulting in changes in the structure of the gut microbiota. Beck et al. [25] showed that the Alpha diversity in very preterm infants supplemented with probiotics was significantly higher than that in the placebo group (P = 0.035). Hui et al. [27] also found that Lactobacillus rhamnosus and B. animalis Bb-12 were significantly increased in fecal samples of very premature infants receiving probiotic supplementation, and probiotic supplementation also affected the Alpha and Beta diversity of the gut microbiota. Notably, some scholars have confirmed [28] that children with attention deficit, hyperactivity, and autism spectrum disorders have a lower diversity of gut microbiota. Animal studies [29] have reported that changes in gut microbiota diversity can predict neural outcomes. These results suggest that early intervention with probiotics can improve the diversity and richness of the gut microbiota in very premature infants and may have potential benefits in improving related diseases and neurological outcomes.
Previous studies reported that probiotics can effectively prevent LOS and NEC in very premature infants. Deshmukh et al. [30] conducted a meta-analysis of 30 clinical studies, and the results showed that probiotics could significantly reduce the incidence of NEC (4.2% in the probiotic group vs. 6.9% in the placebo group, odds ratio: 0.60; 95% confidence interval: 0.50–0.73; P < 0.000). A systematic review and network meta-analysis [31] included 106 trials involving 25,840 preterm infants. It revealed that moderate to high-certainty evidence demonstrated an association between multistrain probiotics and reduction in all-cause mortality; these interventions were also associated with the best effectiveness for other key outcomes, including severe necrotizing enterocolitis, culture-proven sepsis, feeding intolerance, time to reach full enteral feeding, and hospitalization duration. The prediction of gut microbiota KEGG function in this study showed that early intervention with probiotics can help prevent infectious diseases and digestive system diseases and further confirmed that probiotics can effectively prevent LOS and NEC in very preterm infants; however, the specific mechanism remains unclear.
The gut microbiota interacts with and is critical in the intestinal barrier [32]. Studies have shown that serum Zonulin protein and FC can be used as indicators to evaluate intestinal barrier function and permeability [33; 34]. In this study, it was found that the abundance of Bifidobacterium in the observation group was highly expressed on day 14 and day 28, 12.8 times that of the control group (P = 0.000) and 2.6 times that of the control group (P = 0.041), respectively, and the levels of serum Zonulin protein and FC were significantly lower than those of the control group (P < 0.000). The authors speculated that probiotics might improve gut barrier function by altering the composition of gut bacterial communities and reducing serum Zonulin and FC levels. This finding is consistent with those of the previous studies. A European randomized controlled trial [35] showed that infants who received probiotics and prebiotics had lower mean FC concentrations, lower gut inflammation, and a predominance of the Bifidobacterium genus in the gut microbiota during the intervention period, and this microbial advantage quickly disappeared when these supplemental formulations were discontinued. Another study by the Austrian Medical Biological Research Center showed that the FC and serum Zonulin levels of 10 patients with psoriatic arthritis who received probiotics containing Lactobacillus and Bifidobacterium for 12 weeks were significantly reduced, suggesting that the intake of probiotics may improve intestinal permeability and disease activity by altering the structure of the gut microbiota [36]. Kaczmarczyk et al. [16] found that the composition of the gut microbiota and related metabolic potential changed in the first 2 years of life and were associated with Zonulin and FC levels. Therefore, we speculated that probiotics may affect serum Zonulin and FC levels and improve intestinal barrier function by altering gut microbiota characteristics.
In conclusion, early intervention with a probiotic (B. animalis Bb-12) can change the composition of the gut microbiota, regulate serum Zonulin protein and FC levels, and improve intestinal barrier function. This study provides a theoretical basis for early intervention in preterm infants using probiotics and the implications of its potential mechanisms.