Raymond Kiu

Background: The detrimental impact of alcohol consumption on the gut microbiome is well-established. However, less is known about how alcohol exposure during pregnancy affects the maternal gut and vaginal microbiota, or how these microbial changes relate to subsequent infant diagnosis of fetal alcohol spectrum disorder (FASD). We therefore investigated associations between self-reported alcohol use during pregnancy, infant FASD diagnosis, and maternal gut and vaginal microbiota. Methods: Fecal samples (n = 207) and vaginal swabs (n = 28) from pregnant participants recruited through antenatal clinics in the Western Cape Province of South Africa were profiled by 16S rRNA V1-V2 amplicon sequencing. Maternal alcohol use was assessed using Alcohol Use Disorder Identification Test (AUDIT) scores, and FASD was diagnosed in infants using revised Institute of Medicine criteria. Microbial diversity, taxonomic profiles and PICRUSt2 -predicted functional pathways were analyzed using vegan , phyloseq and MaAsLin3 . Results: Maternal AUDIT scores were negatively associated with maternal gut microbiota richness, Shannon, and Inverse Simpson diversity ( p < 0.05). Observed richness was also reduced in participants whose infants were diagnosed with FASD ( p = 0.046). Gut microbiota community structure was not significantly associated with alcohol use or infant FASD diagnosis. However, taxonomic and functional analysis suggested gram-positive taxa depletion with alcohol use and FASD diagnosis, and impaired one-carbon metabolism among participants with infants diagnosed with FASD. Vaginal microbiota diversity and composition were not associated with alcohol use or infant FASD diagnostic group. Conclusions: This is the first human study to investigate the maternal gut and vaginal microbiota in relation to alcohol use during pregnancy and infant FASD outcomes. Our findings suggest that alcohol use is associated with disruption of the maternal gut microbiota, with potential implications for FASD development in exposed infants. Further investigation of alcohol-associated maternal microbial disturbances may inform microbiota-targeted strategies to improve maternal and infant health linked to alcohol use.

Infant gut microbiota development involves frequent colonization by Enterobacteriaceae , particularly Escherichia coli , yet their ecological role in healthy infants is unclear. Here, we analyse longitudinal stool samples from healthy, term-born, breastfed infants (n = 41) and related mothers (n = 30) using shotgun metagenomics and novel computational approaches. Strain-resolved profiling indicates that Bifidobacterium species are frequently shared within families, whereas E. coli derive from external sources, but often persist within individuals. Despite differing ecological strategies, these genera co-exist and share evolutionary adaptations related to lactose acquisition in the infant gut. In vitro, we demonstrate that interactions between E. coli and Bifidobacterium bifidum are mutualistic in co-culture, where E. coli supplies cysteine to its auxotrophic partner, facilitating cooperative degradation of 2′-fucosyllactose, the predominant human milk oligosaccharide. In turn, the liberated monosaccharides sustain E. coli growth, highlighting a cooperative cross-feeding interaction that may contribute to regulating E. coli abundance within the infant host.

Infant gut microbiome development is strongly impacted by breastmilk and human milk oligosaccharides (HMOs), which can protect preterm infants against pathologies including necrotizing enterocolitis. HMO metabolism in bifidobacteria is well characterized and linked to health outcomes, but the scope of HMO-utilizing species remains unclear. Here, using a combination of genomics, proteomics and metabolomics, we show that Clostridium species isolated from preterm infants (born at Clostridium perfringens lacking the toxin perfringolysin O (PfoA), metabolized HMOs. Clostridium species produced beneficial metabolites including short-chain fatty acids and tryptophan catabolites at higher quantities than Bifidobacterium species in vitro. Cell-free supernatant from C. perfringens was non-toxic to colonic cell lines, promoted the growth of commensal bifidobacteria and inhibited growth of pathobionts isolated from the preterm infant gut in vitro. It also suppressed inflammation in preterm-derived intestinal organoids. These findings expand our understanding of HMO-metabolizing microbes and suggest that pfoA ⁻ C. perfringens strains could contribute to healthy infant gut development.

Bifidobacterium species and strains are key members of the human gut microbiota, appearing soon after birth and persisting into adulthood. Resistant starch is an important dietary substrate for adult-associated bifidobacteria, where its fermentation supports host health. However, less is known about how different starch structures interact with bifidobacteria. Here we show that growth kinetics and gene expression differ depending on starch structure. Using detailed growth assays, genomics, and metabolomic analyses, bifidobacterial starch hydrolysis capabilities were closely associated with their CAZyme profiles. In one isolate of Bifidobacterium globosum, we identified a gene cluster encoding three multi-functional amylase enzymes complemented by several starch-binding modules, the genes and proteins of which were significantly upregulated in response to starch. Homologs of genes in the cluster were found in the genomes of bifidobacterial isolates from weaning infants providing insights into their role in the maturation process of the microbiota. Uncovering mechanisms of metabolic interaction between starch structures and bifidobacteria underscores the importance of this ecological function and potential health implications.

Introduction - Patients with advanced chronic liver disease (ACLD) and underlying cirrhosis frequently require repeated courses of antimicrobial therapy, with both the frequency and spectrum of antimicrobial exposure increasing alongside disease progression. In this population, impaired immune function and increased intestinal barrier dysfunction contribute to a heightened susceptibility of multidrug-resistant bacterial infections. Objective - To comprehensively characterise the gastrointestinal microbiome and antimicrobial resistance gene (ARG) landscape across the clinical spectrum of ACLD, and to identify microbial and resistome signatures associated with disease severity. Design - We employed long-read metagenomic sequencing (Oxford Nanopore Technologies) to profile the gastrointestinal microbiome and resistome across distinct ACLD stages: acute-on-chronic liver failure (ACLF), decompensated cirrhosis (DC), and stable cirrhosis, compared to healthy controls. Results - ACLF patients had pronounced levels of Enterococcus faecium, with six samples out of 28 showing over 95% relative abundance, suggesting its potential as a bacterial biomarker for advanced cirrhosis. We reconstructed 28 high-quality MAGs of E. faecium from cirrhosis patients, 17 of which originated from ACLF cases. This dominance of E. faecium correlated with substantially reduced microbial diversity and a marked depletion of key commensal taxa, including Blautia, Akkermansia, Faecalibacterium, and Bifidobacterium. Resistome analysis revealed significant enrichment of clinically relevant ARGs in DC and ACLF, including those conferring resistance to aminoglycosides, beta-lactams, and glycopeptides, correlating with prior antimicrobial exposure. Conclusion - Long-read metagenomics enables high-resolution characterisation of microbial and resistome dynamics across ACLD severities. By capturing taxonomic shifts, functional potential, and ARG enrichment, this approach provides valuable insights into microbiome trajectories linked with disease severity, informing mechanistic research and potential clinical interventions.

Preterm infants (300 metagenome-assembled genomes and obtained ~90 isolate genomes via targeted culturomics, allowing strain-level analysis. We also assessed ex vivo horizontal gene transfer (HGT) capacity of multidrug-resistant (MDR) Enterococcus using neonatal gut models. Here we show that probiotic supplementation significantly reduced antibiotic resistance gene prevalence, MDR pathogen load, and restored typical early-life microbiota profile. However, persistent MDR pathogens like Enterococcus , with high HGT potential, underscore the need for continued surveillance. Our findings underscore the complex interplay between antibiotics, probiotics, and HGT in shaping the neonatal microbiome and support further research into probiotics for antimicrobial stewardship in preterm populations.

Staphylococcus haemolyticus is an important cause of sepsis in preterm infants, with gut colonization being recognized as a risk factor for infection. To better understand the diversity of S. haemolyticus among preterm infants, we generated genome sequences of S. haemolyticus strains (n = 140) from 44 stool samples of 22 preterm infants from four hospitals in England. Core genome phylogenetic analyses, incorporating 126 publicly available S. haemolyticus genome sequences, showed that 85/140 (60.1%) of the isolates, from three different hospitals, formed a clonal group with 78/85 (91.7%) strains having Multi-Locus Sequence Type (ST) 49. Antibiotic resistance genes were prevalent in the genomes. There was a strong association between the presence of mecA and phenotypic resistance to oxacillin, and the aacA-aphD gene and phenotypic resistance to gentamicin. While mecA was near-ubiquitous, none of the strains from the preterm infant cohort had a complete Staphylococcal Cassette Chromosome mec (SCCmec) element. The aacA-aphD gene was associated with the transposon Tn4001 in multiple chromosomal and plasmid contexts. Our data suggest the existence of a distinct sub-population of S. haemolyticus that has adapted to colonize the gut of preterm infants, and widespread horizontal gene transfer and recombination among this frequent colonizer of the preterm infant gut.

Gut microbiome development is strongly impacted by breastmilk and human milk oligosaccharides (HMOs), with short- and long-term health implications. HMO metabolism is best characterised within bifidobacteria, but the full range of other HMO-utilising species remains unknown. This study examined HMO-utilising bacteria that colonise preterm infants (born Clostridium perfringens , and three other Clostridium species, metabolise HMOs, producing short chain fatty acids, tryptophan catabolites, and other metabolites known to improve host health. C. perfringens inhibited pathobiont growth and supressed inflammation in preterm-derived intestinal organoids. These findings suggest a previously unrecognised role for C. perfringens , specifically those lacking the perfringolysin O gene, in promoting healthy gut development during infancy.

Background: The Kasai portoenterostomy (KPE) aims to re-establish bile flow in biliary atresia (BA); however, BA remains the commonest indication for liver transplantation in pediatrics. Gut microbiota-host interplay is increasingly associated with outcomes in chronic liver disease. This study characterized fecal microbiota and fatty acid metabolites in BA. Methods: Fecal samples were prospectively collected in newly diagnosed BA infants (n = 55) before and after KPE. Age-matched healthy control (n = 19) and cholestatic control (n = 21) fecal samples were collected. Fecal 16S rRNA gene amplicon sequencing for gut microbiota and gas chromatography for fecal fatty acids was performed. Results: Increased abundance of Enterococcus in pre-KPE BA and cholestatic control infants, compared to healthy infants, was demonstrated. At the early post-KPE time points, increased alpha diversity was revealed in BA versus healthy cohorts. A lower relative abundance of Bifidobacterium and increased Enterococcus, Clostridium, Fusobacterium, and Pseudomonas was seen in infants with BA. Fecal acetate was reduced, and fecal butyrate and propionate were elevated in early post-KPE BA infants. Higher post-KPE alpha diversity was associated with nonfavorable clinical outcomes (6-month jaundice and liver transplantation). A higher relative abundance of post-KPE Streptococcus and Fusobacterium and a lower relative abundance of Dorea, Blautia, and Oscillospira were associated with nonfavorable clinical outcomes. Blautia inversely correlated to liver disease severity, and Bifidobacterium inversely correlated to fibrosis biomarkers. Bifidobacterium abundance was significantly lower in infants experiencing cholangitis within 6 months after KPE. Conclusions: Increased diversity, enrichment of pathogenic, and depletion of beneficial microbiota early post-KPE are all factors associated with nonfavorable BA outcomes. Manipulation of gut microbiota in the early postsurgical period could provide therapeutic potential.

Bifidobacterium species and strains are key members of the gut microbiota, appearing soon after birth and persisting into adulthood. Resistant starch is an important dietary substrate for adult-associated bifidobacteria, where its fermentation supports host health. However, little is known about how different starch structures interact with bifidobacteria across various ages and ecological niches. To address this, we carried out detailed growth kinetics screening of Bifidobacterium reference strains and unique isolates from breast-fed infants, testing their metabolic interaction with a variety of starch structures. ¹ H NMR metabolomics as well as analysis of CAZyme profiles from genomes were generated for each Bifidobacterium -starch combination. For a subset of resistant starch-utilising isolates, we integrated multi-omics approaches to attain further mechanistic interaction insights. Our results revealed that bifidobacterial starch hydrolysis capabilities are closely associated with their CAZyme profiles and appear to be connected to the niche they occupy. Notably, in one isolate of Bifidobacterium pseudolongum , we identified a novel gene cluster containing three multi-functional amylase enzymes complemented by several starch binding modules which were significantly upregulated in response to resistant starch. This gene cluster was also found in the genomes of bifidobacterial isolates from weaning infants and adults. These findings provide new insights into their participation in the maturation process of the infant gut microbiota. Uncovering mechanisms of metabolic interaction between starch structures and bifidobacteria underscores the importance of this ecological function and potential health implications.