Abstract

Background

   Recent studies suggest a role of gut microbiota in the development of
   neonatal hyperbilirubinemia, with bifidobacteria showing promise in
   alleviating symptoms. However, uncertainties persist regarding gut
   bifidobacterial species composition and their effects on bilirubin
   metabolism. Therefore, the study investigated the association between
   the gut microbiota and neonatal hyperbilirubinemia, assessing the
   potential and underlying mechanisms of Bifidobacterium in managing the
   condition.

Results

   Compared to the high-risk group (requiring clinical intervention),
   low-risk neonates (designated as controls without therapeutic needed)
   demonstrated a higher abundance of breastfeeding-associated
   Bifidobacterium species, including Bifidobacterium longum subsp.
   infantis, B. bifidum, and B. breve. In an experimental neonatal
   hyperbilirubinemia rat model, these Bifidobacterium species’ protective
   effects were demonstrated by reducing serum bilirubin levels,
   maintaining growth and development, and improving neurobehavioral
   reflexes. These benefits are associated with improved liver and
   intestinal barrier functions and reduced enterohepatic circulation of
   bilirubin. Among the various treatment groups, B. longum subsp.
   infantis exhibited the most potent effect, followed by B. bifidum and
   B. breve. Intestinal metabolism analysis revealed that the levels of
   arachidonic acid and docosahexaenoic acid were increased and negatively
   correlated with bilirubin levels. These findings were further confirmed
   in the neonatal cohort study. Direct supplementation of these
   metabolites into the colon of neonatal rats improved disease
   phenotypes. In addition, both in vitro and in vivo experiments
   demonstrated the specific inhibitory effect of arachidonic acid and
   docosahexaenoic acid on β-glucuronidase enzyme activity. Notably, both
   in vitro intestinal fermentation models and genomic analyses
   demonstrated that the three Bifidobacterium species cannot synthesize
   these unsaturated fatty acids due to the lack of related genes.
   However, data suggested that these species might promote the
   accumulation of arachidonic acid and docosahexaenoic acid by regulating
   the gut microbiota’s structure and function. Moreover, these species
   show species-specific genomic distribution patterns, which might
   influence the gut microbiota's α-linolenic and linoleic acid metabolism
   pathways, thereby affecting bilirubin levels.

Conclusions

   The results indicate that Bifidobacterium species associated with
   breastfeeding can prevent neonatal hyperbilirubinemia by regulating the
   gut microbiota–α-linolenic and linoleic acid metabolism–enterohepatic
   circulation axis, increasing gut metabolites arachidonic acid and
   docosahexaenoic acid, and reducing the enterohepatic circulation of
   bilirubin.
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   Video Abstract

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s40168-025-02190-y.

   Keywords: Neonatal hyperbilirubinemia, Bilirubin metabolism,
   Bifidobacterium, Gut microbiota, α-linolenic acid and linoleic acid
   metabolic pathway, Arachidonic acid, Docosahexaenoic acid,
   β-glucuronidase

Introduction

   Neonatal jaundice is a prevalent clinical condition affecting
   approximately 60% of full-term infants and 80% of preterm infants
   [[39]1]. While most jaundice cases are benign, vigilant monitoring is
   crucial to prevent progression to severe hyperbilirubinemia or, in rare
   cases, bilirubin encephalopathy or kernicterus [[40]2]. The
   significance of neonatal hyperbilirubinemia has been underscored by
   leading healthcare policy research groups, including the World Health
   Organization’s Child Health Epidemiology Reference Group and the Global
   Burden of Disease Collaborators [[41]3]. Neonatal bilirubin mainly
   comes from the breakdown of hemoglobin in red blood cells [[42]1].
   After being converted by heme oxygenase, it becomes unconjugated
   bilirubin (UCB), which is liposoluble. UCB binds to serum albumin for
   transport to the liver. In the liver, UCB is catalyzed by
   UDP-glucuronosyltransferase (UGT) 1A1 to form water-soluble conjugated
   bilirubin (CB), which is then excreted into the intestine with bile. In
   the enterohepatic circulation, about 10–20% of CB is hydrolyzed back to
   UCB by intestinal β-glucuronidase (β-GD) and reabsorbed into the
   circulation through the intestinal mucosa. Newborns are prone to UCB
   accumulation due to insufficient UGT1A1 activity in the liver and
   active enterohepatic circulation [[43]4, [44]5]. The lipid-soluble
   nature of UCB enables its penetration through the blood–brain barrier,
   where it may deposit in the basal ganglia and cause kernicterus [[45]1,
   [46]2]. Preterm and low-birth-weight infants face more significant
   neurotoxic risks due to their underdeveloped blood–brain barriers
   [[47]1, [48]2].

   Since the 1960 s, the gut microbial community has been recognized as a
   key player in bilirubin degradation [[49]1]. A recent study has
   identified BilR, a bilirubin reductase produced by gut microbiota,
   which converts bilirubin into urobilinogen [[50]6]. While prevalent in
   healthy adults, BilR is relatively rare in neonates and individuals
   with inflammatory bowel disease [[51]6]. The finding highlights the
   role of gut microbiota in bilirubin metabolism and underscores the
   importance of the gut-liver axis in maintaining bilirubin homeostasis.
   Further research has corroborated the gut microbiota’s role in
   bilirubin metabolism, with disruptions evident in germ-free animals or
   those subjected to antibiotics, resulting in decreased fecal
   urobilinogen production and increased serum bilirubin [[52]7].
   Additional evidence suggests an inverse relationship between intestinal
   bifidobacteria abundance and neonatal hyperbilirubinemia risk [[53]8,
   [54]9]. Studies have shown that bifidobacterial probiotics, such
   as"bifid-triple viable"(Bifico^®), may synergistically enhance
   therapeutic outcomes for hyperbilirubinemia when used with oral
   medications, traditional Chinese herbal medicine, or phototherapy
   [[55]10, [56]11]. Hence, administering specific bifidobacteria strains
   is a safe and effective strategy to alleviate neonatal
   hyperbilirubinemia symptoms.

   Our understanding of how these beneficial gut bacterial species and
   metabolites reduce neonatal hyperbilirubinemia risk remains limited.
   The most extensively studied metabolites are short-chain fatty acids
   (SCFAs) produced by gut microbiota, including Bifidobacterium [[57]12].
   A recent collaborative study provided significant evidence of a
   positive correlation between resistance to jaundice from viral
   infections and enrichment of butyric acid-producing taxa like
   Bacteroidetes and Clostridia, coupled with increased
   glutamate/glutamine expression [[58]13]. However, the specific
   mechanisms by which Bifidobacterium influences neonatal
   hyperbilirubinemia remain poorly understood. Critical knowledge gaps
   persist regarding the relationship between gut Bifidobacterium species
   composition and hyperbilirubinemia risk, as well as the specific
   Bifidobacterium species that might mitigate this risk [[59]7]. These
   limitations hinder the clinical application of Bifidobacterium in
   managing neonatal hyperbilirubinemia.

   This study first explored the relationship between neonatal gut
   microbiota colonization patterns and hyperbilirubinemia risk through
   stratified analysis. Meanwhile, we validated three bifidobacteria
   species associated with breastfeeding for their capacity to modulate
   bilirubin levels using a neonatal rat model of experimental
   hyperbilirubinemia. Additionally, we elucidated the role and mechanism
   of differential metabolites arachidonic acid and docosahexaenoic acid
   in alleviating bilirubin metabolism disorders through colon-targeted
   delivery. Furthermore, in vitro intestinal simulation fermentation and
   comparative genomic analysis were conducted to analyze how
   bifidobacteria enrich these differential metabolites in the gut of
   newborn hosts. The study enhances our understanding of gut microbiota
   characteristics associated with neonatal hyperbilirubinemia risk and
   proposes probiotic interventions to decrease the incidence and risk of
   this condition in neonates.

Materials and methods

Participants and sample collection

   The study is an observational study that adhered to the Helsinki
   Declaration’s principles, was approved by the Medical Ethics Committee
   of Bo’ai Hospital, Zhongshan (KY-2022–010-09), and was registered with
   the Chinese Clinical Trial Registry (Registration Number:
   ChiCTR2200064858). Informed consent was obtained from the parents of
   the neonates. The study involved 72 neonates, with samples collected at
   Bo’ai Hospital in Zhongshan, China, from October to December 2022.
   Infants were selected based on the following inclusion criteria: aged
   48 to 144 h (the peak period for neonatal jaundice), born at 35 weeks’
   gestational age or more, with a birth weight of 2500 g or above, and
   delivered vaginally (Table S1). Additionally, after birth, these
   infants had not received any treatments, including antibiotics,
   phototherapy, exchange transfusion, or probiotics. Exclusion criteria
   included known factors causing pathological jaundice, such as
   infection, glucose-6-phosphate dehydrogenase deficiency, autoimmune
   hemolysis, cholestasis, hypoglycemia, polycythemia, hypothermia, and
   perinatal asphyxia [[60]14].

   Venous blood samples from participating infants were collected by
   experienced neonatologists using disposable vacuum blood collection
   tubes; 1 ml of blood was obtained per draw. The infants’ fecal samples
   were collected in sterile tubes, refrigerated, and immediately
   transported to the laboratory for storage at – 80 °C. We assessed the
   risk of neonatal hyperbilirubinemia using the hour-specific bilirubin
   nomogram developed by Bhutani et al., as outlined in the American
   Academy of Pediatrics'"Management of Hyperbilirubinemia in the Newborn
   Infant 35 or More Weeks of Gestation"[[61]15]. Data processing involved
   classifying infants into the high-risk (HR) group when total bilirubin
   levels exceeded the 95th percentile (typically requiring close
   monitoring and treatment due to high bilirubin levels), into the
   medium-risk (MR) group when exceeding the 75th percentile, and into the
   low-risk (LR) group when below the 75th percentile (considered the
   normal group; no treatment indicated). The specific details are shown
   in Table S2.

Bacterial strain isolation and culture

   As previously described in references [[62]16, [63]17], to minimize