Rimke R. de Kroon

Probiotics may reduce the risk of necrotizing enterocolitis (NEC), late-onset sepsis (LOS), and mortality in preterm infants. However, their efficacy in extremely preterm infants (gestational age < 28 weeks) is still regularly questioned due to limited data and safety concerns. In Dutch Neonatal Intensive Care Units (NICUs), a three-strain probiotic product (ProPrems®; Bifidobacterium infantis Bb-02, Bifidobacterium lactis BB-12, and S treptococcus thermophilus TH-4) was implemented as standard of care from 2021 onwards. This study evaluates the clinical effects of these probiotics in extremely preterm infants. Pre- and postimplementation cohorts of extremely preterm infants from 6 Dutch NICUs (2018–2023) were compared. Clinical data from the first 29 days of life were collected. Incidence of NEC (modified Bell’s stage ≥ IIA), LOS, and mortality were compared with logistic regression; growth velocity with linear regression; and time to full enteral feeds (tFEF) with survival analyses. In total, 481 extremely preterm infants (212 pre-, 269 post-implementation) were included. After implementation, the adjusted odds ratio was 0.49 (0.25–0.99; p = 0.049) for NEC; 0.87 (0.59–1.28; p = 0.49) for LOS; and 0.67 (0.35–1.27; p = 0.22) for all-cause mortality. Furthermore, tFEF decreased by 2 days ( p < 0.001) and probiotic use was associated with an increase in growth velocity of 1.8 g/kg/day ( p < 0.001). One infant (0.4%) developed probiotic-associated sepsis. Conclusion : This multicenter pre-postimplementation study showed that multi-strain probiotic administration was associated with lower NEC incidence, shorter tFEF, and greater growth velocity in extremely preterm infants during the first four weeks of life. Probiotic-associated sepsis was rare. These results support continued use of probiotics in this vulnerable population. What is Known: • Certain probiotic strains effectively reduce incidence of necrotizing enterocolitis (NEC) in preterm infants, but the efficacy and safety for extremely preterm infants (GA < 28 weeks) remain under debate. What is New: • This study shows that a multi-strain probiotic supplement was associated with lower NEC incidence, shorter time to full enteral feeds, and greater early postnatal growth in a Dutch multicenter cohort of extremely preterm infants. • The incidence of probiotic-associated sepsis in extremely preterm infants was very low in our cohort with just 1 case (0.4%), consistent with the rare occurrence reported previously.

Intestinal bacterial translocation to the bloodstream is a route of infection for late-onset sepsis (LOS) in preterm infants, highlighting the potential of fecal microbiota profiling for early risk stratification. We aimed to identify and validate LOS-specific gut microbiota signatures. Fifty-eight preterm infants (gestational age < 30 weeks) with blood culture-proven LOS (excluding coagulase-negative staphylococci) were matched to controls (1:1) across three cohorts (Discovery (DC) n = 18; Validation 1 and 2; VC1 n = 12, VC2 n = 28). Fecal samples collected up to 10 days before LOS onset underwent 16S rRNA gene sequencing. Microbial composition, diversity, and discriminatory taxa were compared across LOS subgroups. Random Forest (RF) models were trained in DC and validated in VC1/VC2. Microbiota variation was largely explained by LOS pathogen (R² = 17%, P < 0.001). Infants with non-staphylococcal and E. coli-LOS showed a temporal increase in relative abundance of Escherichia/Shigella. The RF model distinguishing E. coli-LOS from controls displayed the highest discriminatory performance (AUC = 0.99/0.78/0.61 for DC/VC1/VC2) compared to non-staphylococcal LOS (AUC = 0.96/0.46/0.41). Our findings demonstrate profound microbiota shifts preceding E. coli-LOS, with higher discriminatory ability compared to non-staphylococcal-LOS. While pathogen-specific microbiota-based risk stratification may offer added clinical value, reduced validation performance highlights the limited generalizability and underscores the need for future research before clinical translation.

Introduction: Fecal microbiome and metabolome alterations precede late-onset sepsis (LOS) in preterm infants, but clinical determinants of fecal amino acid (AA) composition and association with LOS development remain poorly understood. Objectives: This study assessed the early-life determinants of fecal AA composition and AA profiles prior to clinical LOS onset to improve pathophysiological understanding. Methods: Infants (< 28 weeks’ gestation) with non-staphylococcal LOS were included in a discovery (n = 12) and validation cohort (n = 8), each matched 1:1 to non-LOS controls (n = 20), based on gestational and postnatal age. Using targeted liquid chromatography-tandem mass-spectrometry, this study identified early-life determinants of fecal AAs, analyzing fecal samples collected at week 1 to 4 in controls. Additionally, AA profiles prior to LOS onset were assessed to improve pathophysiological understanding, analyzing samples at t0 and t-3 days from LOS-affected infants and controls. Results: The most common LOS pathogens were Escherichia coli (n = 8), Serratia species (n = 4), and Streptococcus agalactiae (n = 3). Postnatal age, full-enteral feeding status, probiotic administration, and delivery mode significantly influenced fecal AAs. In the discovery cohort, threonine and glutamine were significantly decreased in affected infants (median [Q1-Q3], threonine: 43.8 [29.3–66.3] vs. 64.5 [48.1–107.3] μmol/L, p = 0.02; glutamine: 19.0 [9.0–38.3] vs. 34.1 [27.0–69.8], p = 0.01), while the validation cohort solely showed a non-significant decrease of threonine. When combining both cohorts, threonine achieved an area under the curve of 0.65 (95%-CI: 0.53–0.76). Conclusions: These findings suggest several clinical characteristics influence fecal AAs, and altered threonine metabolism may contribute to LOS pathophysiology, warranting evaluation of related metabolites and multi-marker approaches in future studies.

Children born extremely preterm (EP, < 28 weeks gestation) are at increased risk of lifestyle-related morbidities. This study characterized parent-reported lifestyle behaviours in EP-born children at 2 years’ corrected age (CA) and compared them with a Dutch reference population. In a multicentre prospective cohort study, parents completed the FLY-Kids lifestyle screening questionnaire (2 years’ CA), which assessed parental satisfaction and adherence to age-specific recommendations for diet, eating habits, physical activity, screen time and sleep. Outcomes were compared with a Dutch reference sample of 2-year-olds, and associations were examined using linear regression. Of 269 eligible children, 239 parents responded. After exclusions ( n = 63), 176 children (74%) were enrolled, of whom 115 (65%) completed the questionnaire (14 twin-pairs). Compared with the Dutch reference population ( n = 73), lifestyle recommendations were more often unmet in EP children (mean = 3.92 vs. 3.29 items, p = 0.01). Interestingly, a larger proportion of EP children exceeded the recommended sleep duration (≥ 14 h) ( p < 0.001), while other lifestyle items were similar. In both populations, > 30% of children failed to meet the recommendations for seven out of nine items. Higher socioeconomic status ( p = 0.016) and having at least one foreign-born parent ( p = 0.034) were associated with lower parental satisfaction. Bronchopulmonary dysplasia diagnosis was associated with greater adherence to recommendations ( p < 0.001), while being underweight ( p = 0.029) associated with more unmet recommendations. Conclusion : Despite longer sleep duration, lifestyle behaviours in EP children at 2 years’ CA largely resemble those of the general population. However, poor adherence to lifestyle recommendations highlight the need for targeted interventions to reduce lifestyle-associated morbidities in EP children. What is Known: • Healthy lifestyle behaviours in early childhood are essential for long-term health outcomes. • Extremely preterm (EP)–born children have an elevated risk of lifestyle-related morbidities. However, knowledge on lifestyle behaviours during toddlerhood in this population remains limited. What is New: • Characterization of lifestyle behaviours in this specific group of EP-born children at 2 years’ corrected age offers novel insights. • Except for longer sleep duration, lifestyle behaviours largely resemble the general population. However, over 30% of children in both EP and term populations fail to meet recommendations for 7 of the 9 lifestyle items included in the FLY-Kids tool.

Invasive candidiasis poses a serious risk to preterm infants, due to its rapidly progressive and severe clinical course, resulting in considerable mortality and long-term morbidity. Therefore, the aim of the current study was to assess the clinical characteristics of invasive candidiasis in very preterm infants to increase awareness among clinicians. A multicenter cohort study database was screened for infants, born in one of ten Neonatal Intensive Care Units (NICUs) in the Netherlands and Belgium between October 2014 and May 2025, with blood- and/or cerebrospinal fluid culture-proven invasive candidiasis (gestational age < 30 weeks) in the first 29 days of life. Clinical data were retrospectively collected. Out of 2.824 infants, 24 were diagnosed with invasive candidiasis (0.8%), most frequently caused by Candida albicans (83%). Affected infants demonstrated distinct clinical features: extreme prematurity (mean 25.7 weeks ± 9 days), low birth weight (mean 827 ± 198 g), vaginal delivery (88%), and sepsis and/or gastrointestinal disease prior to clinical onset (46%). In 58%, initiation of antifungal treatment was delayed. The disease course was generally severe with end-organ disseminated candidiasis (33%), need for invasive ventilation (58%), cardiorespiratory support (42%), and red blood cell and/or platelet transfusion (71% and 33%). Both C-reactive protein and platelet count at diagnosis were associated with fatal outcome ( p = 0.040 and p = 0.010, respectively). Conclusion : In infants with distinct clinical features, clinicians should maintain a high index of suspicion, particularly in NICUs with a higher incidence of Candida colonization and/or infection. Our findings underline the need for a rapid diagnostic test to reduce treatment delays and improve clinical outcomes. What is known: • Invasive candidiasis poses a risk to preterm infants due to its rapidly progressive disease course, high mortality, and long-term morbidity. • A critical need exists to enhance vigilance among clinicians, enabling timely diagnosis, and initiation of targeted treatment. What is new: • Affected infants are characterized by shared clinical features and substantial disease burden. • Frequent vaginal delivery in affected infants suggests that colonization and infection may result from vertical transmission, offering opportunities for early prevention.

Gut microbiota profiling shows potential for improving care in the neonatal intensive care unit (NICU). However, common techniques, including 16S rRNA gene and metagenomic sequencing, have limited bedside applicability. The IS‐pro microbiota assay provides species‐level abundances within 5 h. We aimed to optimize the taxa annotation for preterm infants (phase 1), compare its findings to 16S sequencing on the genus level (phase 2), and apply the assay in a preterm cohort (phase 3). 1445 fecal samples from 479 preterm infants (24–30 weeks gestation) across 10 NICUs were analyzed with IS‐pro. For phase 1 (optimization), IS‐pro amplicons of 32 fecal samples were additionally analyzed with nanopore sequencing to expand the IS‐pro matching database. For phase 2 (comparison), 41 samples were compared to 16S sequencing. In phase 3 (application), the optimized IS‐pro assay was applied to the total cohort. Following phase 1, a mean relative abundance of 82.5% was successfully annotated. In phase 2, IS‐pro showed high concordance with 16S sequencing, with a strong positive correlation between the two techniques (Pearson's correlation coefficient: 0.77, SD 0.24). In phase 3, IS‐pro analysis of the full cohort revealed Staphylococcus, Klebsiella, Enterococcus, Escherichia‐Shigella , and Streptococcus as the predominant genera in the first 4 weeks of life. Our findings demonstrate that the IS‐pro microbiota assay effectively detects and quantifies key bacterial taxa in fecal samples of preterm infants, with outcomes highly concordant with 16S sequencing. Unlike traditional techniques, IS‐pro is a rapid tool, illustrating its potential for clinical practice. Future studies should explore its applications in the NICU.

Background: The skin-to-blood route is traditionally considered the main pathway in Candida late-onset sepsis (LOS) development in preterm infants. However, emerging evidence suggests that the gut also serves as a source of infection. We aimed to characterize fecal mycobiota and microbiota profiles preceding onset of Candida LOS to assess the role of the preterm gut microbiome in disease development. Methods: This multicenter case-control study included very preterm infants (<30 weeks of gestation) with Candida LOS. Each case was matched to nonaffected controls by gestational and postnatal age, hospital site, and/or cumulative antibiotic exposure prior to day of LOS onset (t = 0). Fecal samples collected at t = 0 and the 5 preceding days were analyzed by ITS1 and 16S RNA sequencing. Microbial amplicon yields, composition, and interkingdom correlations were assessed. Results: Of 2397 screened infants, fecal samples were available for 8 of 19 infants with Candida LOS. In these 8 cases, the ITS/16S amplicon yield ratio was increased (P < .001), and the relative abundance of fecal Candida albicans correlated positively with fungal amplicon yield (ρ = 0.71, adjusted P = .005), suggesting increased absolute abundance up to 5 days before onset. Additionally, bacterial yields were significantly lower (P = .02) and α-diversity significantly decreased (P = .012) when compared to the controls. Conclusions: Increased fecal C albicans preceded Candida LOS onset, implicating the preterm gut as a potential source of infection. Reduced bacterial yields and diversity suggest ecological alterations that may facilitate Candida pathogenicity in the preterm gut. These findings support further research into gut-derived Candida LOS and the potential for microbiota-targeted prevention strategies.

Early diagnosis and treatment of late-onset sepsis (LOS) is crucial for survival, but challenging. Intestinal microbiota and metabolome alterations precede the clinical onset of LOS, and the preterm gut is considered an important source of bacterial pathogens. Fecal volatile organic compounds (VOCs), formed by physiologic and pathophysiologic metabolic processes in the preterm gut, reflect a complex interplay between the human host, the environment, and microbiota. Disease-associated fecal VOCs can be detected with an array of devices with various potential for the development of a point-of-care test (POCT) for preclinical LOS detection. While characteristic VOCs for common LOS pathogens have been described, their VOC profiles often overlap with other pathogens due to similarities in metabolic pathways, hampering the construction of species-specific profiles. Clinical studies have, however, successfully discriminated LOS patients from healthy individuals using fecal VOC analysis with the highest predictive value for Gram-negative pathogens. This review discusses the current advancements in the development of a non-invasive fecal VOC-based POCT for early diagnosis of LOS, which may potentially provide opportunities for early intervention and targeted treatment and could improve clinical neonatal outcomes. Identification of confounding variables impacting VOC synthesis, selection of an optimal detection device, and development of standardized sampling protocols will allow for the development of a novel POCT in the near future.