Preterm neonates often exhibit gut dysbiosis among other gastrointestinal problems. Gut dysbiosis in preterm neonates is caused by many disadvantageous factors such as Cesarean delivery, nonbreast feeding, exposure to broad spectrum antibiotics and prolonged hospital stay with multiple harsh environmental exposures in the neonatal intensive care unit (NICU) [1-3]. Consequently, gut microbiota of preterm neonates typically has aberrant microbial colonization (i.e., reduced diversity) with notable numbers of pathogenic bacteria such as the Enterobacteriaceae which are Gram-negative facultative anaerobes (i.e., Escherichia coli, Klebsiella pneumoniae) and a lower abundance of commensal obligate anaerobes such as Bifidobacterium, Lactobacillus, and Bacteroides [4], the aforementioned microbiota condition can be defined as gut dysbiosis [5]. Intestinal dysbiosis in preterm neonates has been linked to necrotizing enterocolitis (NEC) [6-8]. Thus, preterm neonates are one of the most vulnerable populations to encounter detrimental gastrointestinal problems, most notably NEC [1]. Fecal microbiota analysis of preterm neonates with NEC demonstrated an increased abundance of Enterobacteriaceae and Clostridiaceae and a decrease of Bifidobacteriaceae, Firmicutes, and Bacteroidetes [1,3].. Besides gut dysbiosis, impaired maturation of gastrointestinal tract as well as the underdevelopment of the gut immune system make it difficult for the preterm neonates to tolerate optimal enteral feeding and undergo optimal and sustainable growth [2,9].

In contrast to gut dysbiosis, healthy gut microbial colonization in neonates also known as gut eubiosis, has been shown as a pivotal role in maintaining balanced intestinal immunity, nutrient digestion and absorption, intestinal motility, and energy utilization [1]. Intestinal eubiosis is typically observed in term, vaginally delivered, exclusively breastfed infants [3,5,10]. The proposed healthy gut microbiota is characterized by an abundance in Gram-positive Firmicutes such as Staphylococcus, Bifidobacterium, and Lactobacillus with the ability to produce glycosidases necessary to digest human milk oligosaccharides (HMOs) [3]. Probiotics supplementation in the early life of preterm neonates might reverse or modify the gut dysbiosis to resemble those of term neonates with gut eubiosis.

Probiotics are live microorganisms that, when administered in adequate amounts, confer a healthy benefit to the host [11]. Early supplementation of probiotics, either multistrain or single-strain, to preterm neonates aims to alter gut microbiota environment by colonizing the immature gut with commensal bacteria which are naturally present in the presumably eubiotic environment of exclusively breastfed term newborns [9,12]. When the gastrointestinal tract is colonized by these bacteria derived from the probiotics, studies have shown that the preterm neonates are less likely to develop late-onset sepsis and NEC [1,3,13]. However, the beneficial mechanisms of probiotics remain largely unknown but are thought to involve changes in intestinal permeability, enhanced mucosal IgA responses to foreign antigens and increased production of anti-inflammatory cytokines [14]. Promising beneficial effects of probiotics have been shown in various clinical trials in the prevention of NEC [14-19], late-onset sepsis [15,16], and reduction in mortality [14,15,17,19]. However, many other previous studies in this area demonstrated inconsistent results, in which some studies did not show clinical benefits of probiotics supplementation in preterm neonates. Two meta-analyses concluded that only specific strains of probiotics such as various strains of Bifidobacterium spp. yielded promising clinical benefits while others did not [16,18]. The 2020 European guideline for using probiotics in preterm neonates only provides a conditional recommendation on administering specific strains of probiotics in the prevention of NEC [20].

A dual-strain probiotics consisting of Bifidobacterium bifidum/Lactobacillus acidophillus (BB/LA) has been shown in neonates to increase the abundance of Bifidobacterium spp. and Lactobacillus spp. with a reduction of Enterobacteriaceae, compared with placebo/the control group [12,21]. The use of probiotics BB/LA has increased among preterm newborns in many NICUs across Europe with good safety profiles [22,23] and has been shown to significantly reduce the risk of NEC, nosocomial bacteremia, sepsis, and death [24-26]. It is well known that breast milk feeding reduces NEC risk in preterm neonates [27,28]. Probiotics has a synergistic effect among the breastfed neonates in altering the gut microbiota [21,29] and preventing NEC [18,23]. A reasonable explanation would be that breast milk contains prebiotic HMOs which act as an important metabolic source of growth for the Bifidobacterium [11,25]. Nowadays many NICUs encourage breast milk as a primary source of enteral feeding in the preterm neonates, we believe that BB/LA would yield advantageous gut microbiota profile for the predominantly breastfed preterm neonates.

Probiotics BB/LA administration in the neonatal period has been shown to maintain the colonization of Bifidobacterium after discontinuing it during the infancy period [21], indicating the sustainable beneficial effects. According to previously mentioned studies, we hypothesized that preterm neonates generally exhibit gut dysbiosis earlier in life, which is characterized by high levels of Enterobacteriaceae and Clostridiacieae and relatively low levels of Bifidobacterium and Lactobacillus. With the aid of probiotics BB/LA, the gut dysbiosis might be reversed or attenuated. Since there are only a handful of randomized controlled studies on the prevention of gut dysbiosis by BB/LA, therefore we aim to compare gut microbial profiling of the preterm infants who receive in-hospital probiotics BB/LA and those who did not.

We enrolled preterm newborns who were born between July 2022 and September 2023. This clinical trial was reviewed and approved by the Institutional Review Board of Faculty of Medicine Ramathibodi Hospital (MURA 2022/153) and registered in the Thai Clinical Trials Registry (TCTR20220829005). This study was carried out with strict adherence to the World Medical Association’s Declaration of Helsinki. Written informed consent was obtained from the parents or guardians of all participants. The inclusion criteria were as follows: preterm newborns with a gestational age (GA) <33 weeks or birth weight <1,500 grams (i.e., very low birth weight). The exclusion criteria were cyanotic congenital heart disease, chromosomal or gastrointestinal tract anomalies, or enteral feeding was withheld for more than 14 consecutive days. The enrolled infants were randomized into the probiotics group (PG) and control group (CG).

Preterm newborns in PG received BB/LA (Infloran, Laboratorio Farmaceutico SIT Mede, Italy). One capsule of BB/LA (250 mg) contains 1 billion colony forming unit (CFU) lyophilized Bifidobacterium bifidum and 1 billion CFU lyophilized Lactobacillus acidophilus. After having been fed breast milk for one day without complications, one capsule of BB/LA was administered once daily via dissolving in 1–2 mL of sterile water prepared by neonatal registered nurses, then tube-fed before feeding milk. BB/LA preparations were freshly prepared right before administering to the infants. Throughout the trial, BB/LA was kept in a 4°C temperature-controlled refrigerator, separated from the milk preparation station to avoid cross-contamination with CG. BB/LA was administered until postmenstrual age (PMA) of 35 weeks and continued until the infants were discharged. In CG, besides a standard neonatal care similar to the PG, preterm newborns did not receive BB/LA during the study. The feeding protocol strongly supported the use of breast milk as the first choice in all studied infants. However, when there was insufficient breast milk in some infants, pasteurized donor human milk (DHM) would be given after written informed consent was obtained from the parents. Preterm newborns in both groups received similar standardized feeding protocol and medical care without interference from the researchers.

Perinatal and maternal data was obtained by interviewing the mother according to the case record form and from electronic medical records. All preterm newborns would receive empirical antibiotics if there was clinical suspicion of neonatal sepsis. Then, if blood cultures were negative at 48 to 72 hours and the newborns had no signs of sepsis, the antibiotics were discontinued. Enteral feeding was initiated for studied newborns within the first few days of life with maternal breast milk (MBM) or DHM in case of unavailable mother’s breast milk. MBM or DHM was continuously given at least 2 weeks after birth but not more than 4 weeks. After 2 weeks, if there was no MBM, premature formula milk was gradually introduced, alternating with MBM/DHM. At any point, if MBM became available, the infants would receive MBM as the first choice. Full enteral feeding was defined as a total volume of 150 mL/kg/day.

The focused clinical outcomes associated with gut dysbiosis were NEC with modified Bell’s stage ≥2A, late-onset neonatal sepsis, and death. Time to full enteral feeding, duration of hospitalization, body weight at discharge, 2 months and 4 months, respiratory distress syndrome and bronchopulmonary dysplasia were also recorded. Factors altering gut microbiota of preterm newborns such as duration of antibiotics administration, route of delivery and types of feeding were also recorded.

The gut microbiota profiles of meconium samples in preterm newborns during the first few days of life in our study were predominantly colonized with Staphylococcus, which is consistent with previous studies [30-32]. This can be explained by the fact that most of the preterm infants in this study were delivered via Cesarean delivery, thus making the gut microbiome of their early life resembled those of the skin surface of their mothers [3,33,34]. Therefore, the overall profile of gut microbiome in the first days of life was therefore not diverse. In accordance with our hypothesis, we found significant difference in the gut microbiota profile after we administered probiotics BB/LA to the newborns. The gut microbiota demonstrated a more eubiotic environment [3,5,10], with significantly higher abundances of Bifidobacterium and Lactobacillus [31]. Whereas in the CG, the relative abundance of Clostridium senso stricto 1 was significantly higher. In previous studies, Clostridium senso stricto 1 was more abundant preceding the development of NEC in the preterm newborns [35]. Another signal of gut dysbiosis was that Bifidobacterium and Lactobacillus were virtually non-existent in CG at PMA 35 weeks. Lower numbers of Bifidobacteriaceae and higher numbers of Clostridiaceae in the preterm newborns could be a signal of gut dysbiosis [3], which was also associated with NEC [36]. Although not statistically different, probably due to a relatively small study population, preterm newborns in the CG had a slightly higher incidence of NEC stage ≥2A (12% vs. 3% in the PG, Table 2). Our research findings are consistent with the previous studies that showed the ability of probiotics to modulate gut microbiota of preterm newborns into a healthier environment [12,21,29,30,37].

We did not find any differences in alpha diversities between the 2 groups at all 3 time points, which are consistent with a previous study by Chang et al. [30]. Alpha diversity measures the richness of bacteria in each sample, hence at birth, the considerably low alpha diversities in both groups were not unexpected because of similar perinatal characteristics including route of delivery (mostly Cesarean delivery), one of the crucial factors contributing to gut dysbiosis early after birth [1,2]. Despite not statistically different after PMA 35 weeks, the alpha diversity in both groups increased overtime which is consistent with the study by Korpela et al. [32], where the diversity of gut microbiota in preterm newborns increased along with postnatal age. The cause of increased alpha diversity might be different between the neonates in both groups. In the PG, their gut microbiota profile was largely modulated by the BB/LA resulting in the higher abundance of Bifidobacterium and Lactobacillus. Infants in the CG were also exposed to the harsh environment such as NICU setting, antibiotics administration, and formula feeding, thus potentially resulting in a higher number of Clostridium senso stricto 1 [35]. Beta diversity at PMA 35 weeks demonstrated a clear dissimilarity of gut microbiota between the 2 groups, in which PG had a more diverse profile consistent with the study by Chang et al. [30].

From discharged until 4 months of age, infants in both groups relocated to their home environment for several weeks, which likely altered their gut microbiota profiles. We found no difference of alpha and beta diversities between the 2 groups. This might be explained by the fact that they were not exposed to hospital environment for several weeks and most of them received similar type of feeding. Probiotics BB/LA could not maintain gut microbiota diversity after its discontinuation. Surprisingly, gut microbiota in both groups showed a predominance of Bifidobacterium despite discontinuing BB/LA after discharged. In addition, probiotics BB/LA might pertain to the colonization of Bifidobacterium in the gut of former preterm infants [21] until 4 months of age after discontinuation. In our research, most patients after discharged receive half of their intake as breast milk and the other half as formula milk. This might explain that the predominance of Bifidobacterium was enhanced by breast milk that contained HMOs which serve as a prebiotic for Bifidobacterium [11,25]. Unfortunately, we were not able to perform the LefSe analysis to depict a statistical difference of Bifidobacterium between the 2 groups because of the limited number of stool samples.

There were several advantages in this clinical trial. We compared the gut microbiota profiles of preterm newborns in 3 different time points; at birth, at PMA 35 weeks and at age 4 months between the infants who received probiotics BB/LA and infants who did not. This comparison elucidated the characteristics of gut microbiota of preterm newborns which exhibited gut dysbiosis. Our NICU has a unified feeding protocol where all preterm newborns mostly receive breast milk or DHM in early weeks after birth. This research also showed that continuing breast milk after discharge from the hospital helped sustaining the abundance of Bifidobacterium. This result again stresses the importance of breast milk in preterm newborns in improving the chance of maintaining gut eubiosis. However, there were some limitations in this study. The number of infants and stool samples for gut microbiota analysis were relatively small. Therefore, we were not able to demonstrate significant differences of gut microbiome at 4 months of follow-up and did not find clinical significances on the use of probiotics BB/LA such as the prevention of NEC and late-onset neonatal sepsis. Our technique of 16s rRNA sequencing only detected the bacteria at the genus level, so we could not tell whether the Bifidobacterium and Lactobacillus found in the stool samples belonged to the probiotic’s species (BB/LA) or not.

In conclusion, this research showed that probiotics BB/LA modulated gut microbiota of preterm newborns into a more eubiotic environment with diverse commensal bacteria such as Bifidobacterium and Lactobacillus and low pathogenic bacteria such as Clostridium senso stricto 1. Studying the metabolomic profile of preterm gut microbiota affected by probiotics may shed the lights on mechanism of action of probiotics and further explain their benefits on clinical outcome.