Sociodemographic Factors and Intestinal Microbiome Development in Preterm, Very Low Birth Weight Infants.

OBJECTIVE: Preterm very low birth weight (VLBW) infants are at risk for intestinal morbidities and dysbiotic development of the intestinal microbiome. Despite the influence of sociodemographic factors on premature infant health outcomes, whether they shape the intestinal microbiome early in life is not clear. The objective was to explore the associations between race, sex, and socioeconomic status and the intestinal microbiome of VLBW infants during the first 4 weeks of life. STUDY DESIGN: This was a secondary analysis of data from an ongoing randomized trial of 79 infants ≤30 weeks' gestation and ≤1,500 g. Stool samples were collected at week 1 through week 4, frozen to -80°C and analyzed by 16S rRNA sequencing of the V4 region using Illumina MiSeq. Reads were analyzed to measure α and ß diversity as well as relative abundance of bacteria in the intestinal microbiome. RESULTS: Of the 79 infants, 63 had at least one sample available. Twenty-three (37%) of infants were African American, 30 (48%) were male, and 44 (71%) had Medicaid insurance. There were no statistically significant (<0.05) differences in α diversity or ß diversity, and the differential abundance analysis suggests limited patterns of distinction in the intestinal microbiome between non-African American and African American infants, male and female infants, and infants with maternal private or Medicaid insurance. CONCLUSION: Our results suggest race, sex, and socioeconomic status shape colonization of specific microorganisms to a limited extent. Future studies should confirm these findings and determine clinical relevance through further study of differentially abundant microorganisms and additional factors contributing to colonization patterns. KEY POINTS: · Diversity of the gut microbiome was similar between infants of varying race, sex, and socioeconomic status.. · We observed sociodemographic-linked differences in colonization of individual taxa.. · Further study is required to confirm these results and the clinical relevance of these findings..

A Scoping Review of the Oral Microbiome in Preterm Infants.

The purpose of this scoping review was to examine the oral microbiome composition in preterm infants, sampling and collection methods, as well as exposures associated with oral microbiome composition and health implications. We conducted a scoping review of the literature using the Arskey and O'Malley framework. We identified a total of 13 articles which met our inclusion criteria and purpose of this scoping review. Articles included in this review compared the oral microbiome in preterm infants to term infants, examined alterations to the oral microbiome over time, compared the oral microbiome to different body site microbiomes, and explored associations with clinically relevant covariates and outcomes. Exposures associated with the diversity and composition of the oral microbiome in preterm infants included delivery mode, oral feeding, oropharyngeal care, skin-to-skin care, and antibiotics. Day of life and birth weight were also associated with oral microbiome composition. The oral microbiome may be associated with the composition of the tracheal and gut microbiomes, likely due to their proximity. Alpha and beta diversity findings varied across studies as well as the relative abundance of taxa. This is likely due to the different sampling techniques and timing of collection, as well as the wide range of infant clinical characteristics. Multiple factors may influence the composition of the oral microbiome in preterm infants. However, given the heterogeneity of sampling techniques and results within this review, the evidence is not conclusive on the development as well as short- and long-term implications of the oral microbiome in preterm infants and needs to be explored in future research studies. KEY POINTS: · Day of life is a critical factor in oral microbiome development in preterm infants.. · The oral microbiome may be associated with tracheal and gut microbiome colonization.. · Future research should examine sampling methodology for examining the oral microbiome.. · Future research should explore associations with the oral microbiome and adverse health outcomes..

Osmotic stress induces long-term biofilm survival in Liberibacter crescens.

Citrus greening, also known as Huanglongbing (HLB), is a devastating citrus plant disease caused predominantly by Liberibacter asiaticus. While nearly all Liberibacter species remain uncultured, here we used the culturable L. crescens BT-1 as a model to examine physiological changes in response to the variable osmotic conditions and nutrient availability encountered within the citrus host. Similarly, physiological responses to changes in growth temperature and dimethyl sulfoxide concentrations were also examined, due to their use in many of the currently employed therapies to control the spread of HLB. Sublethal heat stress was found to induce the expression of genes related to tryptophan biosynthesis, while repressing the expression of ribosomal proteins. Osmotic stress induces expression of transcriptional regulators involved in expression of extracellular structures, while repressing the biosynthesis of fatty acids and aromatic amino acids. The effects of osmotic stress were further evaluated by quantifying biofilm formation of L. crescens in presence of increasing sucrose concentrations at different stages of biofilm formation, where sucrose-induced osmotic stress delayed initial cell attachment while enhancing long-term biofilm viability. Our findings revealed that exposure to osmotic stress is a significant contributing factor to the long term survival of L. crescens and, possibly, to the pathogenicity of other Liberibacter species.

PrbP modulates biofilm formation in Liberibacter crescens.

In Liberibacter asiaticus, PrbP is a transcriptional regulatory protein involved in survival and persistence during host infection. Tolfenamic acid was previously found to inhibit interactions between PrbP and the promotor region of rplK, resulting in reduced survival of L. asiaticus in the citrus host. In this study, we performed transcriptome analyses to elucidate the PrbP regulon in L. crescens, as it is phylogenetically the closest related species to L. asiaticus that can be grown in laboratory conditions. Chemical inhibition of PrbP with tolfenamic acid revealed that PrbP is involved in the regulation of diverse cellular processes, including stress response, cell motility, cell cycle and biofilm formation. In vitro DNA binding and bacterial two-hybrid assays also suggested that PrbP is a global regulator of multiple transcription factors (RpoH, VisN, PleD, MucR, MocR and CtrA) at both transcriptional and/or post-transcriptional levels. Sub-lethal concentrations of tolfenamic acid significantly reduced the attachment of L. crescens during biofilm formation and decreased long-term persistence in biofilm structures. Overall, our findings show the importance of PrbP in regulating diverse biological processes through direct and indirect interactions with other transcriptional regulators in L. crescens.

Metabolomic Profile of Personalized Donor Human Milk.

Human milk could be considered an active and complex mixture of beneficial bacteria and bioactive compounds. Since pasteurization drastically reduces the microbial content, we recently demonstrated that pasteurized donor human milk (DHM) could be inoculated with different percentages (10% and 30%) of mother's own milk (MOM) to restore the unique live microbiota, resulting in personalized milk (RM10 and RM30, respectively). Pasteurization affects not only the survival of the microbiota but also the concentration of proteins and metabolites, in this study, we performed a comparative metabolomic analysis of the RM10, RM30, MOM and DHM samples to evaluate the impact of microbial restoration on metabolite profiles, where metabolite profiles clustered into four well-defined groups. Comparative analyses of DHM and MOM metabolomes determined that over one thousand features were significantly different. In addition, significant changes in the metabolite concentrations were observed in MOM and RM30 samples after four hours of incubation, while the concentration of metabolites in DHM remained constant, indicating that these changes are related to the microbial expansion. In summary, our analyses indicate that the metabolite profiles of DHM are significantly different from that of MOM, and the profile of MOM may be partially restored in DHM through microbial expansion.

Identification of flavonoids as regulators of YbeY activity in Liberibacter asiaticus.

Liberibacter asiaticus is the prevalent causative pathogen of Huanglongbing or citrus greening disease, which has resulted in a devastating crisis in the citrus industry. A thorough understanding of this pathogen's physiology and mechanisms to control cell survival is critical in the identification of therapeutic targets. YbeY is a highly conserved bacterial RNase that has been implicated in multiple roles. In this study, we evaluated the biochemical characteristics of the L. asiaticus YbeY (CLIBASIA_01560) and assessed its potential as a target for antimicrobials. YbeYLas was characterized as an endoribonuclease with activity on 3' and 5' termini of 16S and 23S rRNAs, and the capacity to suppress the E. coli ΔybeY phenotype. We predicted the YbeYLas protein:ligand interface and subsequently identified a flavone compound, luteolin, as a selective inhibitor. Site-directed mutagenesis was subsequently used to identify key residues involved in the catalytic activity of YbeYLas. Further evaluation of naturally occurring flavonoids in citrus trees indicated that both flavones and flavonols had potent inhibitory effects on YbeYLas . Luteolin was subsequently examined for efficacy against L. asiaticus in Huanglongbing-infected citrus trees, where a significant reduction in L. asiaticus gene expression was observed.

The Ferredoxin-Like Protein FerR Regulates PrbP Activity in Liberibacter asiaticus.

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase ß-subunit and a specific sequence on the promoter region. The constitutive expression of prbP observed upon chemical inactivation of PrbP-DNA interactions in vivo indicated that the expression of prbP was not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved. In silico genome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed. In vitro transcription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activity in vitro The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast to prbP of L. asiaticus (prbPLas), mRNA levels of ferR of L. asiaticus (ferRLas) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbPLas is modulated via interactions with FerRLas The induction of ferRLas expression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals in L. asiaticusIMPORTANCE The rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent, Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important for L. asiaticus survival in the plant host. In this study, we investigated the interactions between PrbP in L. asiaticus (PrbPLas) and a ferredoxin-like protein (FerR) in L. asiaticus, FerRLas We show that the presence of FerR stabilizes and augments the activity of PrbPLas In addition, we demonstrate that the expression of ferR is induced by increases in osmolarity in Liberibacter crescens Altogether, these results suggest that FerRLas and PrbPLas may play important roles in the regulation of gene expression in response to changing environmental signals during L. asiaticus infection in the citrus host.

Purification and partial characterization of LdtP, a cell envelope modifying enzyme in Liberibacter asiaticus.

BACKGROUND: The aggressive spread of Liberibacter asiaticus, a bacterium closely associated with citrus greening, has given rise to an acute crisis in the citrus industry, making it imperative to expand the scientific knowledge base regarding L. asiaticus. Despite several endeavors to culture L. asiaticus, this bacterium has yet to be maintained in axenic culture, rendering identification and analysis of potential treatment targets challenging. Accordingly, a thorough understanding of biological mechanisms involved in the citrus host-microbe relationship is critical as a means of directing the search for future treatment targets. In this study, we evaluate the biochemical characteristics of CLIBASIA_01175, renamed LdtP (L,D-transpeptidase). Surrogate strains were used to evaluate its potential biological significance in gram-negative bacteria. A strain of E. coli carrying quintuple knock-outs of all genes encoding L,D-transpeptidases was utilized to demonstrate the activity of L. asiaticus LdtP. RESULTS: This complementation study demonstrated the periplasmic localization of mature LdtP and provided evidence for the biological role of LdtP in peptidoglycan modification. Further investigation highlighted the role of LdtP as a periplasmic esterase involved in modification of the lipid A moiety of the lipopolysaccharide. This work described, for the first time, an enzyme of the L,D-transpeptidase family with moonlighting enzyme activity directed to the modification of the bacterial cell wall and LPS. CONCLUSIONS: Taken together, the data indicates that LdtP is a novel protein involved in an alternative pathway for modification of the bacterial cell, potentially affording L. asiaticus a means to survive within the host.

Challenges for Managing Candidatus Liberibacter spp. (Huanglongbing Disease Pathogen): Current Control Measures and Future Directions.

Huanglongbing (HLB; "citrus greening" disease) has caused significant damages to the global citrus industry as it has become well established in leading citrus-producing regions and continues to spread worldwide. Insecticidal control has been a critical component of HLB disease management, as there is a direct relationship between vector control and Candidatus Liberibacter spp. (i.e., the HLB pathogen) titer in HLB-infected citrus trees. In recent years, there have been substantial efforts to develop practical strategies for specifically managing Ca. Liberibacter spp.; however, a literature review on the outcomes of such attempts is still lacking. This work summarizes the greenhouse and field studies that have documented the effects and implications of chemical-based treatments (i.e., applications of broad-spectrum antibiotics, small molecule compounds) and nonchemical measures (i.e., applications of plant-beneficial compounds, applications of inorganic fertilizers, biological control, thermotherapy) for phytopathogen control. The ongoing challenges associated with mitigating Ca. Liberibacter spp. populations at the field-scale, such as the seasonality of the phytopathogen and associated HLB disease symptoms, limitations for therapeutics to contact the phytopathogen in planta, adverse impacts of broad-spectrum treatments on plant-beneficial microbiota, and potential implications on public and ecosystem health, are also discussed.

Defining the Core Citrus Leaf- and Root-Associated Microbiota: Factors Associated with Community Structure and Implications for Managing Huanglongbing (Citrus Greening) Disease.

Stable associations between plants and microbes are critical to promoting host health and productivity. The objective of this work was to test the hypothesis that restructuring of the core microbiota may be associated with the progression of huanglongbing (HLB), the devastating citrus disease caused by Liberibacter asiaticus, Liberibacter americanus, and Liberibacter africanus The microbial communities of leaves (n = 94) and roots (n = 79) from citrus trees that varied by HLB symptom severity, cultivar, location, and season/time were characterized with Illumina sequencing of 16S rRNA genes. The taxonomically rich communities contained abundant core members (i.e., detected in at least 95% of the respective leaf or root samples), some overrepresented site-specific members, and a diverse community of low-abundance variable taxa. The composition and diversity of the leaf and root microbiota were strongly associated with HLB symptom severity and location; there was also an association with host cultivar. The relative abundance of Liberibacter spp. among leaf microbiota positively correlated with HLB symptom severity and negatively correlated with alpha diversity, suggesting that community diversity decreases as symptoms progress. Network analysis of the microbial community time series identified a mutually exclusive relationship between Liberibacter spp. and members of the Burkholderiaceae, Micromonosporaceae, and Xanthomonadaceae This work confirmed several previously described plant disease-associated bacteria, as well as identified new potential implications for biological control. Our findings advance the understanding of (i) plant microbiota selection across multiple variables and (ii) changes in (core) community structure that may be a precondition to disease establishment and/or may be associated with symptom progression.IMPORTANCE This study provides a comprehensive overview of the core microbial community within the microbiomes of plant hosts that vary in extent of disease symptom progression. With 16S Illumina sequencing analyses, we not only confirmed previously described bacterial associations with plant health (e.g., potentially beneficial bacteria) but also identified new associations and potential interactions between certain bacteria and an economically important phytopathogen. The importance of core taxa within broader plant-associated microbial communities is discussed.

The transcriptional activator LdtR from 'Candidatus Liberibacter asiaticus' mediates osmotic stress tolerance.

The causal agent of Huanglongbing disease, 'Candidatus Liberibacter asiaticus', is a non-culturable, gram negative, phloem-limited α-proteobacterium. Current methods to control the spread of this disease are still limited to the removal and destruction of infected trees. In this study, we identified and characterized a regulon from 'Ca. L. asiaticus' involved in cell wall remodeling, that contains a member of the MarR family of transcriptional regulators (ldtR), and a predicted L,D-transpeptidase (ldtP). In Sinorhizobium meliloti, mutation of ldtR resulted in morphological changes (shortened rod-type phenotype) and reduced tolerance to osmotic stress. A biochemical approach was taken to identify small molecules that modulate LdtR activity. The LdtR ligands identified by thermal shift assays were validated using DNA binding methods. The biological impact of LdtR inactivation by the small molecules was then examined in Sinorhizobium meliloti and Liberibacter crescens, where a shortened-rod phenotype was induced by growth in presence of the ligands. A new method was also developed to examine the effects of small molecules on the viability of 'Ca. Liberibacter asiaticus', using shoots from HLB-infected orange trees. Decreased expression of ldtRLas and ldtPLas was observed in samples taken from HLB-infected shoots after 6 h of incubation with the LdtR ligands. These results provide strong proof of concept for the use of small molecules that target LdtR, as a potential treatment option for Huanglongbing disease.

Contribution of the Salmonella enterica KdgR Regulon to Persistence of the Pathogen in Vegetable Soft Rots.

During their colonization of plants, human enteric pathogens, such as Salmonella enterica, are known to benefit from interactions with phytopathogens. At least in part, benefits derived by Salmonella from the association with a soft rot caused by Pectobacterium carotovorum were shown to be dependent on Salmonella KdgR, a regulator of genes involved in the uptake and utilization of carbon sources derived from the degradation of plant polymers. A Salmonella kdgR mutant was more fit in soft rots but not in the lesions caused by Xanthomonas spp. and Pseudomonas spp. Bioinformatic, phenotypic, and gene expression analyses demonstrated that the KdgR regulon included genes involved in uptake and metabolism of molecules resulting from pectin degradation as well as those central to the utilization of a number of other carbon sources. Mutant analyses indicated that the Entner-Doudoroff pathway, in part controlled by KdgR, was critical for the persistence within soft rots and likely was responsible for the kdgR phenotype.

A dual role of the transcriptional regulator TstR provides insights into cyanide detoxification in Lactobacillus brevis.

In this study we uncover two genes in Lactobacillus brevis ATCC 367, tstT and tstR, encoding for a rhodanese and a transcriptional regulator involved in cyanide detoxification. TstT (LVIS_0852) belongs to a new class of thiosulphate:cyanide sulphurtransferases. We found that TstR (LVIS_0853) modulates both the expression and the activity of the downstream-encoded tstT. The TstR binding site was identified at -1 to +33, from tstR transcriptional start site. EMSA revealed that sulphite, a product of the reaction catalysed by TstT, improved the interaction between TstR:P(tstR), while Fe(III) disrupted this interaction. Site-directed mutagenesis in TstR identified M64 as a key residue in sulphite recognition, while residues H136-H139-C167-M171 formed a pocket for ferric iron co-ordination. In addition to its role as a transcriptional repressor, TstR is also involved in regulating the thiosulphate:cyanide sulphurtransferase activity of TstT. A threefold increase in TstT activity was observed in the presence of TstR, which was enhanced by the addition of Fe(III). Overexpression of the tstRT operon was found to increase the cyanide tolerance of L. brevis and Escherichia coli. The protein-protein interaction between TstR and TstT described herein represents a novel mechanism for regulation of enzymatic activity by a transcriptional regulator.

Bile promotes Lactobacillus johnsonii N6.2 extracellular vesicle production with conserved immunomodulatory properties.

Recently, Lactobacillus johnsonii N6.2-derived extracellular vesicles (EVs) were shown to reduce apoptosis in human beta cell lines and stimulate insulin secretion in human islets. Our goal was to identify a physiologically relevant environmental condition that induces a hypervesiculation phenotype in L. johnsonii N6.2 and to evaluate if transcriptional changes are involved in this process. Culturing this strain in the presence of 0.2% bovine bile, which mimics a stressor encountered by the bacterium in the small intestine, resulted in approximately a 100-fold increase in EVs relative to cells grown in media without bile. Whole transcriptome analysis of cells grown with bile revealed upregulation of several peptidoglycan hydrolases as well as several genes involved in fatty acid utilization. These results suggest that the hypervesiculation phenotype may be the result of increased cell wall turnover combined with increased accumulation of phospholipids, in agreement with our previous proteomic and lipidomics results. Additionally, EVs isolated from L. johnsonii N6.2 grown in presence of bile maintained their immunomodulatory properties in host-derived ßlox5 pancreatic and THP-1 macrophage cell lines. Our findings suggest that in L. johnsonii N6.2 vesiculogenesis is significantly impacted by the expression of cell wall modifying enzymes and proteins utilized for exogenous fatty acid uptake that are regulated at the transcriptional level. Furthermore, this data suggests that vesiculogenesis could be stimulated in vivo using small molecules thereby maximizing the beneficial interactions between bacteria and their hosts.

Lactobacillus johnsonii N6.2 phospholipids induce immature-like dendritic cells with a migratory-regulatory-like transcriptional signature.

Shifts in the gut microbiota composition, called dysbiosis, have been directly associated with acute and chronic diseases. However, the underlying biological systems connecting gut dysbiosis to systemic inflammatory pathologies are not well understood. Phospholipids (PLs) act as precursors of both, bioactive inflammatory and resolving mediators. Their dysregulation is associated with chronic diseases including cancer. Gut microbial-derived lipids are structurally unique and capable of modulating host's immunity. Lactobacillus johnsonii N6.2 is a Gram-positive gut symbiont with probiotic characteristics. L. johnsonii N6.2 reduces the incidence of autoimmunity in animal models of Type 1 Diabetes and improves general wellness in healthy volunteers by promoting, in part, local and systemic anti-inflammatory responses. By utilizing bioassay-guided fractionation methods with bone marrow-derived dendritic cells (BMDCs), we report here that L. johnsonii N6.2 purified lipids induce a transcriptional signature that resembles that of migratory (mig) DCs. RNAseq-based analysis showed that BMDCs stimulated with L. johnsonii N6.2 total lipids upregulate maturation-mig related genes Cd86, Cd40, Ccr7, Icam1 along with immunoregulatory genes including Itgb8, Nfkbiz, Jag1, Adora2a, IL2ra, Arg1, and Cd274. Quantitative reverse transcription (qRT)-PCR analysis indicated that PLs are the bioactive lipids triggering the BMDCs response. Antibody-blocking of surface Toll-like receptor (TLR)2 resulted in boosted PL-mediated upregulation of pro-inflammatory Il6. Chemical inhibition of the IKKα kinase from the non-canonical NF-κB pathway specifically restricted upregulation of Il6 and Tnf. Phenotypically, PL-stimulated BMDCs displayed an immature like-phenotype with significantly increased surface ICAM-1. This study provides insight into the immunoregulatory capacity of Gram-positive, gut microbial-derived phospholipids on innate immune responses.

Internalization of extracellular vesicles from Lactobacillus johnsonii N6.2 elicit an RNA sensory response in human pancreatic cell lines.

Cells of all domains of life can secrete extracellular vesicles (EV). These secreted vesicles have been indicated as vehicles carrying molecules that facilitate intra- and inter-species interaction. Lactobacillus johnsonii N6.2, a bacterium used in probiotic preparations, has been shown to produce nano-sized EV. In the present work we used L. johnsonii N6.2 EV, concentrated from exosome depleted MRS supernatant, to identify the uptake mechanisms of EV and the impact of the RNA cargo in the EV on the upregulation of the cellular response of ßlox5 human pancreatic cells. Using eukaryotic uptake inhibitors, it was found that EV are internalized by the clathrin/dynamin mediated endocytosis pathway. Further co-localization experiments with the endosome markers RAB5, RAB7 and LAMP1 as well as calcein indicated that EV escape the endosome shortly after RAB7 fusion. Using the expression of the 2',5'-oligoadenylate synthetase (OAS) host pathway, previously identified as targeted by L. johnsonii EV, we found that the host cellular response to the EV are dependent on the integrity of the external components of the EV as well as on the RNA cargo. Global transcriptome analysis was performed on EV and the bacterial whole cell. It was found that the RNA transcripts found within the EV largely represent the most abundantly transcribed genes in the bacterial cells such as those associated with protein synthesis and glycolysis. Further analysis showed an enrichment of smaller size transcripts as well as those encoding for membrane bound or extracellular proteins in L. johnsonii's EV.

Oral Care in Critically Ill Infants and the Potential Effect on Infant Health: An Integrative Review.

BACKGROUND: Critically ill infants admitted to the neonatal intensive care unit are at risk for ventilator-associated pneumonia and abnormal oral colonization. Adherence to evidence-based guidelines for oral care in critically ill adults is associated with improved short- and long-term health outcomes. However, oral care guidelines for critically ill infants admitted to the neonatal intensive care unit have not been established, possibly increasing their risk of ventilator-associated pneumonia and other health complications. OBJECTIVE: To describe and summarize the evidence regarding oral care for critically ill infants admitted to the neonatal intensive care unit and to identify gaps needing further investigation. METHODS: The MEDLINE (through PubMed) and CINAHL databases were searched for observational studies and randomized controlled trials investigating the effect of oral care on oral colonization, ventilator-associated pneumonia, and health outcomes of infants in the neonatal intensive care unit. RESULTS: This review of 5 studies yielded evidence that oral care may promote a more commensal oral and endotracheal tube aspirate microbiome. It may also reduce the risk of ventilator-associated pneumonia and length of stay in the neonatal intensive care unit. However, the paucity of research regarding oral care in this population and differences in oral care procedures, elements used, and timing greatly limit any possible conclusions. CONCLUSIONS: Oral care in critically ill infants may be especially important because of their suppressed immunity and physiological immaturity. Further appropriately powered studies that control for potential covariates, monitor for adverse events, and use recommended definitions of ventilator-associated pneumonia are needed to make clinical recommendations.

Pasteurization of human milk affects the miRNA cargo of EVs decreasing its immunomodulatory activity.

In this report, we evaluated the effect of the pasteurization (P) process of mother's own milk (MOM) on the miRNA content of extracellular vesicles (EVs) and its impact on innate immune responses. Differences in size or particle number were not observed upon pasteurization of MOM (PMOM). However, significant differences were observed in the EV membrane marker CD63 and miRNA profiles. miRNA sequencing identified 33 differentially enriched miRNAs between MOMEV and PMOMEV. These changes correlated with significant decreases in the ability of PMOMEV to modulate IL-8 secretion in intestinal Caco2 cells where only MOMEV were able to decrease IL-8 secretion in presence of TNFα. While EVs from MOMEV and PMOMEV were both able to induce a tolerogenic M2-like phenotype in THP-1 macrophages, a significant decrease in the transcript levels of IL-10 and RNA sensing genes was observed with PMOMEV. Together, our data indicates that pasteurization of MOM impacts the integrity and functionality of MOMEV, decreasing its EVs-mediated immunomodulatory activity. This data provides biomarkers that may be utilized during the optimization of milk processing to preserve its bioactivity.

Identification of food and nutrient components as predictors of Lactobacillus colonization.

A previous double-blind, randomized clinical trial of 42 healthy individuals conducted with Lactobacillus johnsonii N6.2 found that the probiotic's mechanistic tryptophan pathway was significantly modified when the data was stratified based on the individuals' lactic acid bacteria (LAB) stool content. These results suggest that confounding factors such as dietary intake which impact stool LAB content may affect the response to the probiotic treatment. Using dietary intake, serum metabolite, and stool LAB colony forming unit (CFU) data from a previous clinical trial, the relationships between diet, metabolic response, and fecal LAB were assessed. The diets of subject groups with high vs. low CFUs of LAB/g of wet stool differed in their intakes of monounsaturated fatty acids, vegetables, proteins, and dairy. Individuals with high LAB consumed greater amounts of cheese, fermented meats, soy, nuts and seeds, alcoholic beverages, and oils whereas individuals with low LAB consumed higher amounts of tomatoes, starchy vegetables, and poultry. Several dietary variables correlated with LAB counts; positive correlations were determined for nuts and seeds, fish high in N-3 fatty acids, soy, and processed meats, and negative correlations to consumption of vegetables including tomatoes. Using machine learning, predictors of LAB count included cheese, nuts and seeds, fish high in N-3 fatty acids, and erucic acid. Erucic acid alone accurately predicted LAB categorization, and was shown to be utilized as a sole fatty acid source by several Lactobacillus species regardless of their mode of fermentation. Several metabolites were significantly upregulated in each group based on LAB titers, notably polypropylene glycol, caproic acid, pyrazine, and chondroitin sulfate; however, none were correlated with the dietary intake variables. These findings suggest that dietary variables may drive the presence of LAB in the human gastrointestinal tract and potentially impact response to probiotic interventions.

Lactobacillus brevis responds to flavonoids through KaeR, a LysR-type of transcriptional regulator.

The ability of transcription factors to respond to flavonoids as signal molecules was investigated in Lactobacillus brevis. Through in vitro screening of a small library of flavonoids, LVIS1989 (KaeR), a LysR-type transcriptional regulator (LTTR), was identified as responsive to kaempferol. The modulation of KaeR activity by flavonoids was characterized in vivo and in vitro. DNase I footprint assays identified the binding of KaeR at two distinctive sites, one in the intergenic region between LVIS1988 and kaeR (-39 to +2) and another within LVIS1988 (-314 to -353, from kaeR translational start point). EMSA assays revealed that both binding sites are required for KaeR binding in vitro. Furthermore, KaeR-DNA interactions were stabilized by the addition of kaempferol (20 µM). In vivo qRT-PCR experiments performed in L. brevis confirmed that the divergently transcribed genes LVIS1988, LVIS1987 and LVIS1986 and kaeR are upregulated in the presence of kaempferol, indicating the role of KaeR as a transcriptional activator. Transcriptional lacZ fusions using Bacillus subtilis as a surrogate host showed that expression of kaeR and LVIS1988 were induced by the presence of the flavonoid. These results indicate that KaeR belongs to a small and poorly understood group of LTTRs that are positively autoregulated in the presence of a ligand.