Harnessing Microbes: A New Era in Combating Antimicrobial Resistance in Neonatal Care

Understanding the Vulnerability of Premature Infants

Approximately one in ten newborns arrives prematurely, with very-low-birth-weight (VLBW) infants, weighing less than 1500 grams, facing particular fragility. In the Neonatal Intensive Care Unit (NICU), broad-spectrum antibiotics are often essential for survival, yet their administration can disturb the delicate gut microbial ecosystem. This disruption can impede the development of a robust immune system and leave infants susceptible to harmful pathogens. Meanwhile, the global health community, specifically the World Health Organization (WHO), advocates for the use of certain probiotic strains in very preterm infants fed exclusively human milk. This recommendation raises a crucial question: can routine probiotic use counteract the adverse effects of antibiotics? Given the prevalence of multidrug-resistant (MDR) infections in NICUs, comprehending the impact of both probiotics and antibiotics on the 'resistome' (the collective of antibiotic resistance genes) is paramount for guiding safer and more effective therapeutic approaches.

Investigating Microbial Dynamics in a Controlled Clinical Setting

This sub-study, part of the broader Baby-Associated Microbiota of the Intestine (BAMBI) observational cohort, involved 34 VLBW preterm infants born before 33 weeks of gestation. All infants were exclusively fed human milk or donor breast milk. Participants were divided into two groups: one receiving probiotic supplementation (Infloran®, containing Bifidobacterium bifidum and Lactobacillus acidophilus) and a non-supplemented control group. Within each group, some infants received short courses of empirical antibiotics (benzylpenicillin and/or gentamicin for an average of three days), while others did not. Fecal samples were collected weekly for the first three weeks of life. Advanced shotgun metagenomic sequencing was employed to analyze the gut microbiome, enabling the reconstruction of metagenome-assembled genomes and the tracking of bacterial strains. Standardized bioinformatics pipelines were used to determine taxonomic and functional profiles, and antibiotic resistance genes were identified using comprehensive databases. Techniques like Average Nucleotide Identity (ANI) and Multi-Locus Sequence Typing (MLST) helped ascertain genetic relatedness and sequence types. To investigate horizontal gene transfer (HGT), the researchers quantified plasmid replicons and modeled potential transfer events. An ex vivo infant gut experiment further explored the transfer of antibiotic resistance, using preterm-derived Enterococcus faecium donor strains. Statistical analyses, including ordination and group-difference testing, were performed to compare outcomes across cohorts and antibiotic exposure groups.

Key Findings on Probiotic Impact and Resistance Patterns

Over the three-week study period, infants receiving probiotic supplements exhibited a gut microbiome predominantly characterized by Bifidobacterium species, particularly the administered Bifidobacterium bifidum, showing active replication. Conversely, the non-supplemented cohort displayed a higher prevalence of early-life pathobionts such as Klebsiella, Escherichia, Enterococcus, and Staphylococcus. Beneficial infant-specific species, including Bifidobacterium breve and Bifidobacterium longum, emerged earlier and in greater abundance with probiotic intervention, aligning with their capacity to utilize human milk oligosaccharides and provide colonization resistance. Functional pathway analysis revealed diverging profiles between the two groups starting from the second week, mirroring the observed taxonomic differences. The resistome analysis indicated that probiotic-supplemented infants possessed fewer antibiotic resistance genes and fewer classes of resistance compared to their non-supplemented counterparts. While resistance to common antibiotics like aminoglycosides, macrolides-lincosamides-streptogramins, beta-lactams, trimethoprim, and tetracyclines was observed in both groups, fluoroquinolone and colistin resistance were exclusively found in non-supplemented infants. Notably, a colistin resistance gene (mcr-9.1) was detected in one non-supplemented sample, highlighting the silent circulation of critical resistance determinants. Short courses of empirical antibiotics did not drastically alter alpha-diversity but did lead to early shifts towards Klebsiella or Enterococcus dominance, especially in the first week. Correlation analyses demonstrated a clear inverse relationship: higher Bifidobacterium abundance correlated with fewer antibiotic resistance genes, while increased levels of Enterococcus and Staphylococcus were associated with a greater burden of resistance genes. Strain-level analysis confirmed that Enterococcus, Escherichia, Klebsiella, and Staphylococcus harbored the most significant resistance profiles. Remarkably, no Klebsiella or Escherichia genomes from the probiotic group met the definition of multidrug resistance (resistance to three or more antibiotic classes), in stark contrast to 47.6% of Escherichia strains from the non-supplemented group. MLST identified several clinically relevant sequence types, indicating potential nosocomial transmission. Plasmid analysis revealed a higher plasmid load in non-supplemented infants and a weak correlation between plasmid counts and antibiotic resistance gene counts. Furthermore, horizontal gene transfer events were more frequent following antibiotic exposure, suggesting that even brief antibiotic courses can promote the movement of mobile genetic elements. The ex vivo infant gut model provided direct evidence of an aminoglycoside resistance gene transferring via a plasmid from Enterococcus faecium to a recipient, conferring gentamicin resistance. This suggests that while probiotics can shift the gut environment towards beneficial microbes and away from pathobionts rich in resistance genes, they do not completely eliminate the risk of horizontal gene transfer. Therefore, judicious antibiotic stewardship remains crucial in curbing plasmid-mediated resistance in the neonatal unit.

Charting a Course for Healthier Preterm Futures

In essence, this study underscores that providing probiotic supplements to very-low-birth-weight preterm infants fosters a gut microbial community rich in Bifidobacterium, significantly reducing the prevalence of antibiotic resistance genes and limiting the emergence of multidrug-resistant characteristics when compared to infants who do not receive probiotics. Despite these benefits, Enterococcus strains continue to act as a substantial reservoir for resistance. Furthermore, even brief antibiotic treatments can heighten the likelihood of horizontal gene transfer, particularly the plasmid-mediated spread of resistance genes. From a practical standpoint, integrating evidence-based probiotic interventions with stringent antibiotic stewardship and robust infection control measures is vital for safeguarding this vulnerable population. Future research necessitates longer-term follow-up studies and multi-site clinical trials to precisely determine optimal probiotic dosages, duration of supplementation, and the most effective bacterial strains to maximize health benefits while simultaneously mitigating the risks associated with antimicrobial resistance. For families and neonatal care units alike, a cohesive strategy encompassing feeding practices, rigorous hygiene, and thoughtful prescribing will be instrumental in improving the long-term health outcomes for premature infants.