Neonatal Microbiota Research Articles

Intestinal microbiota and neonatal necrotizing enterocolitis

The intestinal microbiota is composed of beneficial bacteria and harmful bacteria in the body as the ecological environment,which is the largest,most complex microecosystem.There is emerging evidence that appropriate colonization process of intestinal microbes contributes to development of intestinal structure and function and maturation of immune system,which determines the risk of intestinal diseases.More and more studies focus on the relationship between intestinal microecology or probiotics and neonatal necrotizing enterocolitis (NEC).This article reviews the composition and function of neonatal intestinal microbiota,the intestinal microflora in the pathogenesis and mechanism of NEC and preventive effects of probiotics on NEC. Key words: Necrotizing enterocolitis; Microecology; Probiotics; Neonate

Probiotic administration in congenital heart disease: a pilot study

ObjectiveInvestigate the impact of probiotic Bifidobacterium longum ssp infantis on the fecal microbiota and plasma cytokines in neonates with congenital heart disease.Study designSixteen infants with congenital heart disease were randomly assigned to receive either B. infantis (4.2 × 109 cfu twice daily) or placebo for 8 weeks. Stool specimens from enrolled infants and from six term infants without heart disease were analyzed for microbial composition. Plasma cytokines were analyzed weekly in the infants with heart disease.ResultsHealthy control infants had increased total bacteria, total Bacteroidetes, and total bifidobacteria compared to the infants with heart disease, but there were no significant differences between the placebo and probiotic groups. Plasma IL10, IFNγ, and IL1β levels were transiently higher in the probiotic group.ConclusionsCongenital heart disease in infants is associated with dysbiosis. Probiotic B. infantis did not significantly alter the fecal microbiota. Alterations in plasma cytokines were inconsistent.

1390 Bacterial Colonization of Preterm Infants: Impact of Biofilm Formation on the Nasogastric Feeding Tubes

BackgroundNowadays, hospitals foment breastfeeding or human donor milk to provide the best feeding option to the preterm infant. This fact may represent a key factor in the initiation and development...

Reduced Diversity of the Intestinal Microbiota During Infancy Is Associated With Increased Risk of Allergic Disease at School Age

H Bisgaard, N Li, K Donnelykke. J Allergy Clin Immunol. 2011;128(3):646–652 To investigate the potential association between the diversity of neonatal intestinal microbiota and the development of atopic disorders in childhood. Four hundred eleven infants born in Copenhagen to mothers with a history of asthma were enrolled from the years 1998 to 2001. Infants had an initial visit at 1 month of age and were subsequently seen for a scheduled visit every 6 months until the age of 6 years. Atopic disease was assessed through examination by doctors at the clinical research unit with support from symptom diaries kept by the parents. Investigators conducted interval testing of peripheral blood eosinophil count, skin prick tests, …

Screening Fecal Enterococci from Greek Healthy Infants for Susceptibility to Antimicrobial Agents

Enterococci are among the first lactic acid bacteria to colonize the neonatal gastrointestinal tract, but they are also characterized as significant nosocomial pathogens. The aim of this study was to investigate the incidence of antibiotic resistance in enterococci isolated from neonates' gut microbiota as well as the presence of genetic determinants encoding for certain antibiotic resistance traits. A total of 263 fecal samples derived from 97 infants were collected on day 4, 30, and 90 after delivery. Enterococcus faecalis was the most frequently identified species (54.6%) followed by E. faecium, while E. casseliflavus/E. flavescens and E. gallinarum were also traced. The isolates were examined for their resistance to 12 antibiotics. Rifampicin resistance was the highest observed (53.2%), followed by resistance to tetracycline (42.0%), erythromycin (35.7%), and vancomycin (11.2%). Multiresistant strains were highly prevalent. Only intrinsic vancomycin resistance (vanC1 and vanC2/C3) was traced. The ermB gene was detected in 49 out of 96 erythromycin-resistant isolates, while tet genes were detected in 51 out of 113 tetracycline-resistant strains, with tet(L) being the most frequently observed. In conclusion, antibiotic-resistant enterococci are already established in the fecal microbiota of healthy neonates, from the first days of an infant's life.

Influence of Maternal Bifidobacteria on the Development of Gut Bifidobacteria in Infants

Intestinal microbiota plays an important role in human health by influencing metabolic activities that result in the creation of energy and absorbable nutrients, a barrier to the colonization of pathogens, and stimulation of the immune system. The development of fecal microbiota in neonates is crucial because those bacteria are the first to colonize the sterile intestine of the neonates and, thus, have a significant effect on the host. Initial colonization is also relevant to the final composition of the permanent microbiota in adults. Bifidobacteria are predominant in the fecal microbiota of infants, and, therefore, they are important to an understanding of how commensal bifidobacteria is established in the intestine of infants. While the mother’s bifidobacteria are considered to significantly influence the infant’s bifidobacteria, it is not clear whether a specific bifidobacterial strain transmits vertically from mother to infant and what factors of the mother before delivery influence the establishment of intestinal bifidobacteria in infants. This review focuses on the impact of maternal bifidobacteria on the development of gut bifidobacteria in the infant and suggests that there is cumulative evidence regarding bifidobacterial transfer from the maternal gut or breast milk to the infant gut.

Transmission of the major skin microbiota, Malassezia, from mother to neonate

Skin surface colonization starts after birth. It is thought that early microbial colonization affects the development of skin immune functions. Although Malassezia is the predominant fungus in the skin microbiota in healthy individuals, the microorganism is associated with atopic dermatitis and seborrheic dermatitis. In the present study, transmission of skin microbiota from mothers to their neonates was elucidated using the Malassezia microbiota as an indicator. Temporal changes in the level of Malassezia colonization of the skin from 27 neonates and mothers were investigated by real-time polymerase chain reaction assay. The genotypes of Malassezia colonizing the neonate and mother were also determined. Malassezia was detected from 89% and 100% of neonate samples on days 0 and 1 after birth, respectively. Subsequently, the level of Malassezia colonization of the neonates increased with time, whereas that of the mothers did not change. The Malassezia diversity of neonates shifted to the adult type by day 30. The genotype of Malassezia colonizing the skin of neonates agreed well with that of Malassezia colonizing the skin of the mothers. Fungal microbiota colonization of neonates began on day 0, and the fungal microbiota of neonates had changed to the adult type by day 30. To our knowledge, this is the first report of a molecular analysis of the fungal microbiota of neonates.

Establishment and development of intestinal microbiota in preterm neonates

Microbial colonization of the infant gut is essential for the development of the intestine and the immune system. The profile of intestinal microbiota in the full-term, vaginally delivered, breast-fed infant is considered as ideally healthy. However, in preterm infants this process is challenging, mainly because of organ immaturity, antibiotics use, and hospital stay. To assist in a proper microbiota development in these infants, a detailed knowledge of the colonization process, and the differences from that of full-term breast-fed infants, is needed. We assessed the establishment of the gut microbiota and its metabolic activity in preterm neonates (n=21) during the first 3months of life and compared it with that of vaginally delivered, exclusively breast-fed full-term infants (n=20) using qualitative and quantitative culture-independent methods. Differences in the gut microbiota composition between both groups were observed. Preterm infants showed higher levels of facultative anaerobic microorganisms and reduced levels of strict anaerobes such as Bifidobacterium, Bacteroides, and Atopobium. Short-chain fatty acids concentrations were lower in preterm infants during the first days of life. Alterations occur in the process of microbiota establishment in preterm infants, indicating the need for intervention strategies to counteract them.

Pyrosequencing‐based Molecular Monitoring of the Intestinal Bacterial Colonization in Preterm Infants

The aim of the study was to investigate the previously unexplored diversity of neonatal intestinal microbiota and monitor early intestinal colonization patterns in Korean preterm infants using high-throughput pyrosequencing technology combined with 16S rDNA-based molecular methods. A total of 46,369 partial 16S rDNA sequences obtained from 30 fecal samples serially taken from 10 very-low-birth weight preterm infants were analyzed. A significant proportion of the molecular species (21.9%) was found to be unclassified. The pathogenic or potentially pathogenic molecular species belonging to the classes Gammaproteobacteria and Bacilli were predominant during the entire observation period. Anaerobic or nonpathogenic molecular species belonging to the class Clostridia (except Clostridium difficile) and phyla Bacteroidetes were ubiquitous even within 72 hours after birth. The proportion of these species increased significantly at 1 month of age. The most ubiquitous and abundant major molecular genera common to all of the postnatal ages were Escherichia, Enterobacter, Enterococcus, Veillonella, Serratia, Staphylococcus, Roseburia, Acinetobacter, Citrobacter, Bacteroides, Faecalibacterium, Blautia, and Streptococcus. The diversity and dynamic nature of intestinal bacterial colonization in very-low-birth weight preterm infants were revealed using pyrosequencing technology. The results of the present pilot study may provide a basis to consider when investigating or interpreting the role of intestinal microbiota in certain preterm infant diseases such as necrotizing enterocolitis or systemic infection.

The Pioneer Gut Microbiota in Human Neonates Vaginally Born at Term—A Pilot Study

The pioneer microbiota of the neonate may affect future actions of the immune system. This study aimed to map the pioneer microbiota in healthy neonates vaginally born at term. A subgroup of neonates born large for GA (LGA) was compared with the neonates appropriate for GA (AGA). Fecal samples were collected, within 48 h after birth, from 79 neonates. Quantitative PCR was used for enumeration of Lactobacillus, a subgroup of Lactobacillus common in the vagina, Bifidobacterium, Enterococcus, Enterobacteriaceae, and the Bacteroides fragilis group. Cloning and sequencing were applied for subgroups of neonates born LGA or AGA. Lactobacillus was detected in all neonates, whereas other bacterial groups were detected only in 14 to 30% of the subjects. The prevalence of Gram-negative Proteobacteria was higher in neonates born LGA, whereas Gram-positive Firmicutes was more prevalent in neonates born AGA (p < 0.001). This study contributed to increased knowledge of the pioneer microbiota and indicates that neonates born LGA had significantly different microbiota compared with those born AGA. As the early microbiota can be important for maturation of the immune system, the outcome from this study may be relevant in the care of pregnant woman and newborns.

Transmission of Intestinal Bifidobacterium longum subsp. longum Strains from Mother to Infant, Determined by Multilocus Sequencing Typing and Amplified Fragment Length Polymorphism

The gastrointestinal tracts of neonates are colonized by bacteria immediately after birth. It has been discussed that the intestinal microbiota of neonates includes strains transferred from the mothers. Although some studies have indicated possible bacterial transfer from the mother to the newborn, this is the first report confirming the transfer of bifidobacteria at the strain level. Here, we investigated the mother-to-infant transmission of Bifidobacterium longum subsp. longum by genotyping bacterial isolates from the feces of mothers before delivery and of their infants after delivery. Two hundred seven isolates from 8 pairs of mothers and infants were discriminated by multilocus sequencing typing (MLST) and amplified fragment length polymorphism (AFLP) analysis. By both methods, 11 strains of B. longum subsp. longum were found to be monophyletic for the feces of the mother and her infant. This finding confirms that these strains were transferred from the intestine of the mother to that of the infant. These strains were found in the first feces (meconium) of the infant and in the feces at days 3, 7, 30, and 90 after birth, indicating that they stably colonize the infant's intestine immediately after birth. The strains isolated from each family did not belong to clusters derived from any of the other families, suggesting that each mother-infant pair might have unique family-specific strains.

If I'd known I was going to live this long, I'd have taken better care of myself:Antioxidants and micronutrients in CF

Asthma and allergy incidence continue to increase globally. We have made significant strides in treating disease, but it is becoming more apparent that we need to advance our knowledge into the origins of asthmatic disease. Much recent work has indicated that microbiome composition influences immune regulation and that multiple health care factors have driven a loss in microbiome diversity in modern human populations. Evidence is growing of microbiota-driven influences on immune development, asthma susceptibility, and asthma pathogenesis. The focus of this review is to highlight the strides the field has made in characterizing the constituents of the human gastrointestinal microbiota, such as Helicobacter pylori, other members of the neonatal intestinal microbiota, and microbial peptides and metabolites that influence host immunity and immune response to allergens. As we delve further into this field of research, the goal will be to find actionable and clinical interventions to identify at-risk populations earlier to prevent disease onset. Manipulation of the host microbial community during infancy might be an especially promising approach.

Assessment of intestinal microbiota of full-term breast-fed infants from two different geographical locations

The intestinal microbiota in the breast-fed infant is considered as ideally healthy. We assessed the microbiota of breast-fed full-term neonates from two different Spanish locations. Statistically significant geographical differences for different bacterial groups were found, underlining the need to consider and define geographical-related effects on microbiota.

Ex vivo stimulation of neonatal porcine peripheral blood mononuclear cells with oligosaccharides found in human milk

Infant formulas lack the complex mixture of oligosaccharides (HMO) found in human milk, which may be pivotal to the development of the neonatal microbiota and immune system. Few comprehensive analyses of the direct effects of HMO on immune cells from neonates have been undertaken. Herein, peripheral blood mononuclear cells (PBMC) isolated from 10 and 23 day old (do) sow‐reared pigs were stimulated with HMO (2FL, 3FL, 3SL, 6SL, SL, LNFPIII, LnNT, sLeX and HMO isolated from human milk [iHMO]) for 72h. T cell phenotype, cytokine production and proliferation were measured by flow cytometry, ELISA and 3‐H thymidine incorporation. Of the HMO tested, none had significant effects on any parameters examined. However, developmental differences were found. PBMC from 23do pigs produced more cytokines, had a higher proportion of CD8+CD3+ T cells and proliferated less than those from 10do pigs. PBMC from 23do pigs made more IL‐10 in response to iHMO, PHA or PHA+iHMO than PBMC from 10do pigs. Only PHA or PHA+iHMO stimulation induced IL‐4 secretion, and 10do pigs produced nearly twice as much IL‐4 as 23do pigs. Lastly, PBMC from 10do pigs proliferated 6–10 times more in response to PHA stimulation than did PBMC from 23do pigs. In summary, while appropriate developmental responses were observed, direct effects of HMO on porcine PBMC phenotype, cytokine production or proliferation ex vivo were not detected. (Supported by Abbott Nutrition)

Denaturing gradient gel electrophoresis of neonatal intestinal microbiota in relation to the development of asthma

BackgroundThe extended 'hygiene hypothesis' suggests that the initial composition of the infant gut microbiota is a key determinant in the development of atopic disease. Several studies have demonstrated that the microbiota of allergic and non-allergic infants are different even before the development of symptoms, with a critical time window during the first 6 months of life. The aim of the study was to investigate the association between early intestinal colonisation and the development of asthma in the first 3 years of life using DGGE (denaturing gradient gel electrophoresis).MethodsIn a prospective birth cohort, 110 children were classified according to the API (Asthma Predictive Index). A positive index included wheezing during the first three years of life combined with eczema in the child in the first years of life or with a parental history of asthma. A fecal sample was taken at the age of 3 weeks and analysed with DGGE using universal and genus specific primers.ResultsThe Asthma Predictive Index was positive in 24/110 (22%) of the children. Using universal V3 primers a band corresponding to a Clostridum coccoides XIVa species was significantly associated with a positive API. A Bacteroides fragilis subgroup band was also significantly associated with a positive API. A final DGGE model, including both bands, allowed correct classification of 73% (80/110) of the cases.ConclusionFecal colonisation at age 3 weeks with either a Bacteroides fragilis subgroup or a Clostridium coccoides subcluster XIVa species is an early indicator of possible asthma later in life. These findings need to be confirmed in a new longitudinal follow-up study.

Lactobacillus casei Abundance Is Associated with Profound Shifts in the Infant Gut Microbiome

Colonization of the infant gut by microorganisms over the first year of life is crucial for development of a balanced immune response. Early alterations in the gastrointestinal microbiota of neonates has been linked with subsequent development of asthma and atopy in older children. Here we describe high-resolution culture-independent analysis of stool samples from 6-month old infants fed daily supplements of Lactobacillus casei subsp. Rhamnosus (LGG) or placebo in a double-blind, randomized Trial of Infant Probiotic Supplementation (TIPS). Bacterial community composition was examined using a high-density microarray, the 16S rRNA PhyloChip, and the microbial assemblages of infants with either high or low LGG abundance were compared. Communities with high abundance of LGG exhibited promotion of phylogenetically clustered taxa including a number of other known probiotic species, and were significantly more even in their distribution of community members. Ecologically, these aspects are characteristic of communities that are more resistant to perturbation and outgrowth of pathogens. PhyloChip analysis also permitted identification of taxa negatively correlated with LGG abundance that have previously been associated with atopy, as well as those positively correlated that may prove useful alternative targets for investigation as alternative probiotic species. From these findings we hypothesize that a key mechanism for the protective effect of LGG supplementation on subsequent development of allergic disease is through promotion of a stable, even, and functionally redundant infant gastrointestinal community.

Application of denaturing high-performance liquid chromatography for intestinal microbiota analysis of newborns

Neonatal microbiota development seems to play a key role in the early origins of health and disease. However, the analysis of this complex ecosystem is still difficult. The aim of this work was to investigate the feasibility of a new technique, denaturing high-performance liquid chromatography (dHPLC), to analyze newborn intestinal microbiota using genomic approaches. Eleven neonates were recruited among patients admitted for intestinal surgery to the neonatal intensive care unit. Preoperative samplings were obtained in each case. Three methodologies were compared for each sample: (i) dHPLC, (ii) temporal temperature gradient gel electrophoresis (TTGE), and (iii) conventional culture techniques. All samples were poorly colonized. In three samples, the microbiota was detected only with the dHPLC technique. Results obtained with culture and TTGE could be found with dHPLC. The results suggest that neonatal applications of the dHPLC technique, especially for gut microbiota analysis, appear to be a sensitive and promising analytical technique.

Optimized PCR-Temporal Temperature Gel Electrophoresis compared to cultivation to assess diversity of gut microbiota in neonates

Temporal Temperature Gel Electrophoresis of amplified 16S rRNA gene sequences (16S rDNA PCR-TTGE) constitutes a culture-independent molecular method used to study bacterial communities. All the technical steps are crucial for quality and exhaustiveness of the results obtained by such approach. Careful optimization of the protocols used is ideally needed for each ecosystem studied. We present here the strategy used to construct an optimized protocol for a 16S rDNA PCR-TTGE-based analysis of gut microflora in neonates. Improvement of the different steps, i.e. total DNA extraction, amplification in terms of efficiency and reduction of heteroduplex formation, TTGE migration conditions and bacterial identification from TTGE patterns, was performed. The optimized protocol was used for the subsequent analysis of 14 stool samples comparatively to a culture-based method. We showed that a specifically designed ladder representative of the diversity of the studied microflora is a useful tool for the identification of bacterial taxa despite biases inherent to 16S rRNA genes, including intra-genomic heterogeneity. Cultivation and PCR-TTGE gave congruent results but cultivation was more efficient for the detection of minor populations whereas PCR-TTGE gave a more complete description of the major populations. Finally, we demonstrated the reliability, the detection sensitivity and the convenience of the optimized 16S rDNA PCR-TTGE method compared with cultural approaches for studying the premature neonate gut microbiota.

Influence of antibiotic exposure in the early postnatal period on the development of intestinal microbiota

The influence of antibiotic exposure in the early postnatal period on the development of intestinal microbiota was monitored in 26 infants including five antibiotic-treated (AT) subjects orally administered a broad-spectrum antibiotic for the first 4 days of life and three caesarean-delivered (CD) subjects whose mothers were intravenously injected by the similar type of antibiotics in the same period. The faecal bacterial composition was analysed daily for the first 5 days and monthly for the first 2 months. Terminal restriction fragment length polymorphisms in the AT subjects showed less diversity with the attenuation of the colonization of some bacterial groups, especially in Bifidobacterium and unusual colonization of Enterococcus in the first week than the control antibiotic-free infants (AF, n=18). Quantitative real-time PCR showed overgrowth of enterococci (day 3, P=0.01; day 5, P=0.003; month 1, P=0.01) and arrested growth of Bifidobacterium (day 3, P=0.03) in the AT group. Furthermore, after 1 month, the Enterobacteriaceae population was markedly higher in the AT group than in the AF group (month 1, P=0.02; month 2, P=0.02). CD infants sustained similar, although relatively weaker, alteration in the developing microbiota. These results indicate that antibiotic exposure at the beginning of life greatly influences the development of neonatal intestinal microbiota.

Cesarean Delivery May Affect the Early Biodiversity of Intestinal Bacteria1,

The gastrointestinal tract of neonates becomes colonized immediately after birth with environmental microorganisms, mainly from the mother; strong evidence suggests that the early composition of the microbiota of neonates plays an important role for the postnatal development of the immune system. The present study was designed to evaluate by means of a molecular biology approach the relation between the intestinal ecosystem of the newborn and the mode of delivery. The intestinal bacterial composition on d 3 of life was investigated in 23 infants born by vaginal delivery and in 23 infants delivered by cesarean section. PCR-denaturing gradient gel electrophoresis and PCR-temperature gradient gel electrophoresis have been utilized, together with the specific amplifications for 10 Bifidobacterium species, 3 Ruminococcus species, and Bacteroides. The intestinal microbiota of neonates delivered by cesarean delivery appears to be less diverse, in terms of bacteria species, than the microbiota of vaginally delivered infants. The intestinal microbiota after cesarean delivery is characterized by an absence of Bifidobacteria species. Vaginally delivered neonates, even if they showed individual microbial profiles, were characterized by predominant groups such as B. longum and B. catenulatum. Our data demonstrate that the mode of delivery has a deep impact on the composition of the intestinal microbiota at the very beginning of human life. This study opens the path to further investigations to confirm the link between microbiota composition and immune system development and to identify tools for the modulation of the intestinal microbiota of cesarean-delivered neonates. Additionally, we underline the importance of adequate microbiological tools used to support clinically relevant trials, if intestinal microbiota is considered as a study outcome.