Diet influences community dynamics following vaginal group B streptococcus colonization.

The vaginal microbiota plays a pivotal role in reproductive, sexual, and perinatal health and disease. Unlike the well-established connections between diet, metabolism, and the intestinal microbiota, parallel mechanisms influencing the vaginal microbiota and pathogen colonization remain overlooked. In this study, we combine a mouse model of Streptococcus agalactiae strain COH1 [group B Streptococcus (GBS)] vaginal colonization with a mouse model of pubertal-onset obesity to assess diet as a determinant of vaginal microbiota composition and its role in colonization resistance. We leveraged culture-dependent assessment of GBS clearance and culture-independent, sequencing-based reconstruction of the vaginal microbiota in relation to diet, obesity, glucose tolerance, and microbial dynamics across time scales. Our findings demonstrate that excessive body weight gain and glucose intolerance are not associated with vaginal GBS density or timing of clearance. Diets high in fat and low in soluble fiber are associated with vaginal GBS persistence, and changes in vaginal microbiota structure and composition due to diet contribute to GBS clearance patterns in nonpregnant mice. These findings underscore a critical need for studies on diet as a key determinant of vaginal microbiota composition and its relevance to reproductive and perinatal outcomes.IMPORTANCEThis work sheds light on diet as a key determinant influencing the composition of vaginal microbiota and its involvement in group B Streptococcus (GBS) colonization in a mouse model. This study shows that mice fed diets with different nutritional composition display differences in GBS density and timing of clearance in the female reproductive tract. These findings are particularly significant given clear links between GBS and adverse reproductive and neonatal outcomes, advancing our understanding by identifying critical connections between dietary components, factors originating from the intestinal tract, vaginal microbiota, and reproductive outcomes.

Transforming neonatal care with artificial intelligence: challenges, ethical consideration, and opportunities.

Artificial intelligence (AI) offers tremendous potential to transform neonatology through improved diagnostics, personalized treatments, and earlier prevention of complications. However, there are many challenges to address before AI is ready for clinical practice. This review defines key AI concepts and discusses ethical considerations and implicit biases associated with AI. Next we will review literature examples of AI already being explored in neonatology research and we will suggest future potentials for AI work. Examples discussed in this article include predicting outcomes such as sepsis, optimizing oxygen therapy, and image analysis to detect brain injury and retinopathy of prematurity. Realizing AI's potential necessitates collaboration between diverse stakeholders across the entire process of incorporating AI tools in the NICU to address testability, usability, bias, and transparency. With multi-center and multi-disciplinary collaboration, AI holds tremendous potential to transform the future of neonatology.

Clinical and economic impacts of a modified-observational screening approach to well-appearing infants born to mothers with chorioamnionitis.

OBJECTIVE: Term infants born to mothers with chorioamnionitis are at risk for early-onset sepsis (EOS). We aimed to measure the impact of changing from a categorical to a modified-observational EOS screening approach on NICU admission, antibiotic utilization, and hospitalization costs. STUDY DESIGN: Single-center retrospective pre-post cohort study of full-term infants born to mothers with chorioamnionitis. Primary outcomes included NICU admission, antibiotic utilization, and hospitalization costs. Outcomes were adjusted for demographic variables. Budget-impact analysis was performed using bootstrapping with replication. RESULTS: 380 term infants were included (197 categorical; 183 modified-observational). There was a significant decrease in NICU admission and antibiotic utilization (p < 0.05) in the modified-observational cohort but no significant difference in per-patient total hospitalization costs. Budget-impact analysis suggested a high probability of cost savings. CONCLUSION: A modified-observational approach to evaluating term infants of mothers with chorioamnionitis can reduce NICU admission and unnecessary antibiotic therapy, and may lead to cost-savings.

A group B Streptococcus indexed transposon mutant library to accelerate genetic research on an important perinatal pathogen.

IMPORTANCE: Group B Streptococcus (GBS) is a significant global cause of serious infections, most of which affect pregnant women, newborns, and infants. Studying GBS genetic mutant strains is a valuable approach for learning more about how these infections are caused and is a key step toward developing more effective preventative and treatment strategies. In this resource report, we describe a newly created library of defined GBS genetic mutants, containing over 1,900 genetic variants, each with a unique disruption to its chromosome. An indexed library of this scale is unprecedented in the GBS field; it includes strains with mutations in hundreds of genes whose potential functions in human disease remain unknown. We have made this resource freely available to the broader research community through deposition in a publicly funded bacterial maintenance and distribution repository.

Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects.

Group B Streptococcus (GBS; S. agalactiae ) causes chorioamnionitis, neonatal sepsis, and can also cause disease in healthy or immunocompromised adults. GBS possesses a type II-A CRISPR-Cas9 system, which defends against foreign DNA within the bacterial cell. Several recent publications have shown that GBS Cas9 influences genome-wide transcription through a mechanism uncoupled from its function as a specific, RNA-programmable endonuclease. We examine GBS Cas9 effects on genome-wide transcription through generation of several isogenic variants with specific functional defects. We compare whole-genome RNA-seq from Δ cas9 GBS with a full-length Cas9 gene deletion; dcas9 defective in its ability to cleave DNA but still able to bind to frequently occurring protospacer adjacent motifs; and scas9 that retains its catalytic domains but is unable to bind protospacer adjacent motifs. Comparing scas9 GBS to the other variants, we identify nonspecific protospacer adjacent motif binding as a driver of genome-wide, Cas9 transcriptional effects in GBS. We also show that Cas9 transcriptional effects from nonspecific scanning tend to influence genes involved in bacterial defense and nucleotide or carbohydrate transport and metabolism. While genome-wide transcription effects are detectable by analysis of next-generation sequencing, they do not result in virulence changes in a mouse model of sepsis. We also demonstrate that catalytically inactive dCas9 expressed from the GBS chromosome can be used with a straightforward, plasmid-based, single guide RNA expression system to suppress transcription of specific GBS genes without potentially confounding off-target effects. We anticipate that this system will be useful for study of nonessential and essential gene roles in GBS physiology and pathogenesis.

Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects.

Group B Streptococcus (GBS; S. agalactiae) causes chorioamnionitis, neonatal sepsis, and can also cause disease in healthy or immunocompromised adults. GBS possesses a type II-A CRISPR-Cas9 system, which defends against foreign DNA within the bacterial cell. Several recent publications have shown that GBS Cas9 influences genome-wide transcription through a mechanism uncoupled from its function as a specific, RNA-programmable endonuclease. We examine GBS Cas9 effects on genome-wide transcription through generation of several isogenic variants with specific functional defects. We compare whole-genome RNA-seq from Δcas9 GBS with a full-length Cas9 gene deletion; dcas9 defective in its ability to cleave DNA but still able to bind to frequently occurring protospacer adjacent motifs; and scas9 that retains its catalytic domains but is unable to bind protospacer adjacent motifs. Comparing scas9 GBS to the other variants, we identify nonspecific protospacer adjacent motif binding as a driver of genome-wide, Cas9 transcriptional effects in GBS. We also show that Cas9 transcriptional effects from nonspecific scanning tend to influence genes involved in bacterial defense and nucleotide or carbohydrate transport and metabolism. While genome-wide transcription effects are detectable by analysis of next-generation sequencing, they do not result in virulence changes in a mouse model of sepsis. We also demonstrate that catalytically inactive dCas9 expressed from the GBS chromosome can be used with a straightforward, plasmid-based, single guide RNA expression system to suppress transcription of specific GBS genes without potentially confounding off-target effects. We anticipate that this system will be useful for study of nonessential and essential gene roles in GBS physiology and pathogenesis.

Ventilator-Associated Pneumonia in the Neonatal Intensive Care Unit.

See Bonus NeoBriefs videos and downloadable teaching slides Intubated infants in the NICU are at risk of developing ventilator-associated pneumonia (VAP), a common type of health care-associated infection. The Centers for Disease Control and Prevention developed guidelines for diagnosing VAP in patients younger than 1 year, which include worsening gas exchange, radiographic findings, and at least 3 defined clinical signs of pneumonia. VAP in infants is treated with empiric antibiotics selected based on local resistance patterns and individualized patient data. Many NICUs have implemented prevention bundles in an effort to decrease VAP by ensuring the cleanest environment for intubated neonates (hand hygiene, sterile handling of equipment), positioning of infants to prevent gastric reflux, and constantly reevaluating for extubation readiness. Although these prevention bundle elements are intuitive and generally low risk, none are based on strong research support. This article reviews the epidemiology, pathogenesis, diagnosis, treatment, and prevention of VAP in NICU patients, focusing on recent evidence, highlighting areas of emerging research, and identifying persistent knowledge gaps.

Interpretable prediction of necrotizing enterocolitis from machine learning analysis of premature infant stool microbiota.

BACKGROUND: Necrotizing enterocolitis (NEC) is a common, potentially catastrophic intestinal disease among very low birthweight premature infants. Affecting up to 15% of neonates born weighing less than 1500 g, NEC causes sudden-onset, progressive intestinal inflammation and necrosis, which can lead to significant bowel loss, multi-organ injury, or death. No unifying cause of NEC has been identified, nor is there any reliable biomarker that indicates an individual patient's risk of the disease. Without a way to predict NEC in advance, the current medical strategy involves close clinical monitoring in an effort to treat babies with NEC as quickly as possible before irrecoverable intestinal damage occurs. In this report, we describe a novel machine learning application for generating dynamic, individualized NEC risk scores based on intestinal microbiota data, which can be determined from sequencing bacterial DNA from otherwise discarded infant stool. A central insight that differentiates our work from past efforts was the recognition that disease prediction from stool microbiota represents a specific subtype of machine learning problem known as multiple instance learning (MIL). RESULTS: We used a neural network-based MIL architecture, which we tested on independent datasets from two cohorts encompassing 3595 stool samples from 261 at-risk infants. Our report also introduces a new concept called the "growing bag" analysis, which applies MIL over time, allowing incorporation of past data into each new risk calculation. This approach allowed early, accurate NEC prediction, with a mean sensitivity of 86% and specificity of 90%. True-positive NEC predictions occurred an average of 8 days before disease onset. We also demonstrate that an attention-gated mechanism incorporated into our MIL algorithm permits interpretation of NEC risk, identifying several bacterial taxa that past work has associated with NEC, and potentially pointing the way toward new hypotheses about NEC pathogenesis. Our system is flexible, accepting microbiota data generated from targeted 16S or "shotgun" whole-genome DNA sequencing. It performs well in the setting of common, potentially confounding preterm neonatal clinical events such as perinatal cardiopulmonary depression, antibiotic administration, feeding disruptions, or transitions between breast feeding and formula. CONCLUSIONS: We have developed and validated a robust MIL-based system for NEC prediction from harmlessly collected premature infant stool. While this system was developed for NEC prediction, our MIL approach may also be applicable to other diseases characterized by changes in the human microbiota.

Group B Streptococcus Capsular Serotype Alters Vaginal Colonization Fitness.

BACKGROUND: Group B Streptococcus (GBS) remains a leading cause of infant morbidity and mortality. A candidate vaccine targets 6 GBS serotypes, offering a potential alternative to intrapartum antibiotic prophylaxis to reduce disease burden. However, our understanding of the contributions of specific capsule types to GBS colonization and disease remains limited. METHODS: Using allelic exchange, we generated isogenic GBS strains differing only in the serotype-determining region in 2 genetic backgrounds, including the hypervirulent clonal complex (CC) 17. Using a murine model of vaginal cocolonization, we evaluated the roles of the presence of capsule and of expression of specific capsular types in GBS vaginal colonization fitness independent of other genetic factors. RESULTS: Encapsulated wild-type strains COH1 (CC17, serotype III) and A909 (non-CC17, serotype Ia) outcompeted isogenic acapsular mutants in murine vaginal cocolonization. COH1 wild type outcompeted A909. Notably, expression of type Ia capsule conferred an advantage over type III capsule in both genetic backgrounds. CONCLUSIONS: Specific capsule types may provide an advantage in GBS vaginal colonization in vivo. However, success of certain GBS lineages, including CC17, likely involves both capsule and noncapsule genetic elements. Capsule switching in GBS, a potential outcome of conjugate vaccine programs, may alter colonization fitness or pathogenesis.

Growth and Stress Tolerance Comprise Independent Metabolic Strategies Critical for Staphylococcus aureus Infection.

Staphylococcus aureus is an important pathogen that leads to high morbidity and mortality. Although S. aureus produces many factors important for pathogenesis, few have been validated as playing a role in the pathogenesis of S. aureus pneumonia. To gain a better understanding of the genetic elements required for S. aureus pathogenesis in the airway, we performed an unbiased genome-wide transposon sequencing (Tn-seq) screen in a model of acute murine pneumonia. We identified 136 genes important for bacterial survival during infection, with a high proportion involved in metabolic processes. Phenotyping 80 individual deletion mutants through diverse in vitro and in vivo assays demonstrated that metabolism is linked to several processes, which include biofilm formation, growth, and resistance to host stressors. We further validated the importance of 23 mutations in pneumonia. Multivariate and principal-component analyses identified two key metabolic mechanisms enabling infection in the airway, growth (e.g., the ability to replicate and form biofilms) and resistance to host stresses. As deep validation of these hypotheses, we investigated the role of pyruvate carboxylase, which was important across multiple infection models and confirmed a connection between growth and resistance to host cell killing. Pathogenesis is conventionally understood in terms of the host-pathogen interactions that enable a pathogen to neutralize a host's immune response. We demonstrate with the important bacterial pathogen S. aureus that microbial metabolism influences key traits important for in vivo infection, independent from host immunomodulation. IMPORTANCE Staphylococcus aureus is an important bacterial pathogen that causes significant morbidity and mortality, infecting numerous bodily sites, including the respiratory tract. To identify the bacterial requirements for lung infection, we conducted a genome-wide screen in a mouse model of acute pneumonia. We discovered that metabolic genes were overrepresented in those required for lung infection. In contrast to the conventional view of pathogenesis focusing on immunomodulation, we demonstrate through phenotyping of deletion mutants in several functional assays that replicative ability and tolerance against host defenses form two key metabolic dimensions of bacterial infection. These dimensions are independent for most pathways but are coupled in central carbon metabolism and highlight the critical role of bacterial metabolism in survival against host defenses during infection.

Can we back off using antibiotics in the NICU?

Antibiotics are extensively and inconsistently prescribed in neonatal ICUs, and usage does not correlate with rates of culture positive sepsis. There is mounting data describing the short and long-term adverse effects associated with antibiotic overuse in neonates, including the increased burden of multi-drug resistant organisms. Currently there is considerable variation in antibiotic prescribing practice among neonatologists. Applying the practice of antibiotic stewardship in the NICU is crucial for standardizing antibiotic use and improving outcomes in this population. Several approaches have been proposed to identify neonatal sepsis, with the hope of reducing antibiotic utilization. These strategies all have their limitations, and often include laboratory testing and treatment of well-appearing, non-septic, infants. A conservative "watch and wait" algorithm is suggested as an alternative method for when to initiate antibiotics. This observational approach relies on availability of trained personnel able to examine infants at specified intervals, without delaying antibiotics, should signs of sepsis arise.

Genome-Wide fitness analysis of group B Streptococcus in human amniotic fluid reveals a transcription factor that controls multiple virulence traits.

Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted deletion of the mrvR gene, which had a growth defect in amniotic fluid relative to the wild type parent strain. The mrvR deletion strain also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that while the mutant was able to cause persistent murine vaginal colonization, pregnant mice colonized with the mrvR deletion strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. In a sepsis model the mrvR deletion strain showed significantly decreased lethality. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed RNA-seq on wild type and mrvR deletion GBS strains, which revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting that MrvR may be involved in regulating nucleotide availability.

Human Herpesvirus-6 Meningitis in a Premature Infant with Fevers: A Case and Literature Review.

Human herpesvirus-6 (HHV-6) is a common virus that can cause nearly universal infection in infancy and early childhood. It typically manifests as an acute febrile illness. We describe a case of a premature infant with congenital hydrocephalus secondary to aqueductal stenosis with a ventriculoperitoneal shunt in place who developed intermittent fevers while she was admitted to the neonatal intensive care unit. She was ultimately diagnosed with acute HHV-6 meningitis. In addition to this report, we present a literature review regarding this virus's potential modes of transmission and forms of clinical presentation in the neonatal period.

Multiple Instance Learning for Predicting Necrotizing Enterocolitis in Premature Infants Using Microbiome Data.

Necrotizing enterocolitis (NEC) is a life-threatening intestinal disease that primarily affects preterm infants during their first weeks after birth. Mortality rates associated with NEC are 15-30%, and surviving infants are susceptible to multiple serious, long-term complications. The disease is sporadic and, with currently available tools, unpredictable. We are creating an early warning system that uses stool microbiome features, combined with clinical and demographic information, to identify infants at high risk of developing NEC. Our approach uses a multiple instance learning, neural network-based system that could be used to generate daily or weekly NEC predictions for premature infants. The approach was selected to effectively utilize sparse and weakly annotated datasets characteristic of stool microbiome analysis. Here we describe initial validation of our system, using clinical and microbiome data from a nested case-control study of 161 preterm infants. We show receiver-operator curve areas above 0.9, with 75% of dominant predictive samples for NEC-affected infants identified at least 24 hours prior to disease onset. Our results pave the way for development of a real-time early warning system for NEC using a limited set of basic clinical and demographic details combined with stool microbiome data.

Evaluating Newborns at Risk for Early-Onset Sepsis.

Early-onset sepsis (EOS) is an important cause of neonatal morbidity. Despite extensive study, identifying at-risk newborns remains challenging, especially if they are initially well appearing. Existing official EOS recommendations suggest a conservative approach that likely results in overtreatment of a low-risk population. Recent studies indicate that more precise risk assessment and alternative management strategies could decrease the number of infants exposed to blood draws and antibiotics during evaluations for EOS. This article reviews existing guidelines and provides an overview of the Bayesian sepsis calculator and serial observation as an alternative to laboratory studies and empirical antibiotics.