Laura Moles

Bacterial Diversity in Meconium of Preterm Neonates and Evolution of Their Fecal Microbiota during the First Month of Life

The establishment and succession of bacterial communities in infants may have a profound impact in their health, but information about the composition of meconium microbiota and its evolution in hospitalized preterm infants is scarce. In this context, the objective of this work was to characterize the microbiota of meconium and fecal samples obtained during the first 3 weeks of life from 14 donors using culture and molecular techniques, including DGGE and the Human Intestinal Tract Chip (HITChip) analysis of 16S rRNA amplicons. Culture techniques offer a quantification of cultivable bacteria and allow further study of the isolate, while molecular techniques provide deeper information on bacterial diversity. Culture and HITChip results were very similar but the former showed lower sensitivity. Inter-individual differences were detected in the microbiota profiles although the meconium microbiota was peculiar and distinct from that of fecal samples. Bacilli and other Firmicutes were the main bacteria groups detected in meconium while Proteobacteria dominated in the fecal samples. Culture technique showed that Staphylococcus predominated in meconium and that Enterococcus, together with Gram-negative bacteria such as Escherichia coli, Escherichia fergusonii, Klebsiella pneumoniae and Serratia marcescens, was more abundant in fecal samples. In addition, HITChip results showed the prevalence of bacteria related to Lactobacillus plantarum and Streptococcus mitis in meconium samples whereas those related to Enterococcus, Escherichia coli, Klebsiella pneumoniae and Yersinia predominated in the 3rd week feces. This study highlights that spontaneously-released meconium of preterm neonates contains a specific microbiota that differs from that of feces obtained after the first week of life. Our findings indicate that the presence of Serratia was strongly associated with a higher degree of immaturity and other hospital-related parameters, including antibiotherapy and mechanical ventilation.

Sharing of Bacterial Strains Between Breast Milk and Infant Feces

In previous years, it has been shown that human milk is a potential source of bacteria for the infant gut. The results of this work confirm the presence of the same specific bacterial strains of Bifidobacterium, Lactobacillus, and Staphylococcus in breast milk and infant fecal samples. The identity of bacteria isolated from breast milk and infant feces from 20 mother-infant pairs was investigated at the strain level. DNA from Staphylococcus, Lactobacillus, and Bifidobacterium was detected by qRTi-PCR in nearly all samples analyzed. These samples were cultured on different agar media. One colony representative of each morphology was selected and identified at the species level combining classical tests and molecular techniques (PCR, RAPD, PFGE, and/or MLST genotyping). Breast milk and infant feces from 19 mother-infant pairs shared different Staphylococcus, Lactobacillus, and/or Bifidobacterium species and strains. Significantly, 2 mother-infant pairs shared 4 bacterial strains although most pairs shared 2. These results confirm that breast milk and infant feces from mother-infant pairs share the same strain(s), indicating that breastfeeding could contribute to the bacterial transfer from the mother to the infant and, therefore, to the infant gut colonization.

Preterm infant gut colonization in the neonatal ICU and complete restoration 2 years later

Preterm infants in a neonatal intensive care unit (NICU) are exposed to multidrug-resistant bacteria previously adapted to the hospital environment. The aim of the present study was to characterize the bacterial antibiotic-resistant high-risk lineages colonizing preterm infants during their NICU stay and their persistence in faeces after 2 years. A total of 26 preterm neonates were recruited between October 2009 and June 2010 and provided 144 faecal samples. Milk samples (86 mothers milk, 35 human donor milk and 15 formula milk) were collected at the same time as faecal samples. An additional faecal sample was recovered in 16 infants at the age of 2 years. Samples were plated onto different selective media, and one colony per morphology was selected. Isolates were identified by 16S rDNA nucleotide sequence and MALDI-TOF. Antibiotic susceptibility (agar dilution), genetic diversity (RAPD, PFGE and MLST) and virulence factors (only in enterococcal and staphylococcal isolates) were determined by PCR. A high proportion of antibiotic-resistant high-risk clones was detected in both faecal and milk samples during the NICU admittance. Almost all infants were colonized by Enterococcus faecalis ST64 and Enterococcus faecium ST18 clones, while a wider genetic diversity was observed for the Gram-negative isolates. Multidrug-resistant high-risk clones were not recovered from the faecal samples of the 2-year-olds. In conclusion, the gut of preterm infants admitted to the NICU might be initially colonized by antibiotic-resistant and virulent high-risk lineages, which are later replaced by antibiotic-susceptible community ones.

Bacteriological, biochemical, and immunological properties of colostrum and mature milk from mothers of extremely preterm infants.

Objectives: The objective of this work was to elucidate the influence of extremely premature birth (gestational age 24–27 weeks) on the microbiological, biochemical, and immunological composition of colostrum and mature milk. Methods: A total of 17 colostrum and 34 mature milk samples were provided by the 22 mothers of extremely preterms who participated in this study. Bacterial diversity was assessed by culture-based methods, whereas the concentration of lactose, glucose, and myo-inositol was determined by a gas chromatography procedure. Finally, the concentrations of a wide spectrum of cytokines, chemokines, growth factors, and immunoglobulins were measured using a multiplex system. Results: Bacteria were present in a small percentage of the colostrum and milk samples. Staphylococci, streptococci, and lactobacilli were the main bacterial groups isolated from colostrum, and they could be also isolated, together with enterococci and enterobacteria, from some mature milk samples. The colostrum concentrations of lactose and glucose were significantly lower than those found in mature milk, whereas the contrary was observed in relation to myo-inositol. The concentrations of most cytokines and immunoglobulins in colostrum were higher than in mature milk, and the differences were significant for immunoglobulin G3, immunoglobulin G4, interleukin (IL)-6, interferon-&ggr;, interleukin-4 (IL-4), IL-13, IL-17, macrophage-monocyte chemoattractant protein-1 and macrophage inflammatory protein-1&bgr;. Conclusions: The bacteriological, biochemical, and immunological content of colostrum and mature milk from mothers of extremely preterm infants is particularly valuable for such infants. Efforts have to be made to try that preterm neonates receive milk from their own mothers or from donors matching, as much as possible, the gestational age of the preterm.

Administration of Bifidobacterium breve PS12929 and Lactobacillus salivarius PS12934, Two Strains Isolated from Human Milk, to Very Low and Extremely Low Birth Weight Preterm Infants: A Pilot Study

The preterm infant gut has been described as immature and colonized by an aberrant microbiota. Therefore, the use of probiotics is an attractive practice in hospitals to try to reduce morbidity and mortality in this population. The objective of this pilot study was to elucidate if administration of two probiotic strains isolated from human milk to preterm infants led to their presence in feces. In addition, the evolution of a wide spectrum of immunological compounds, including the inflammatory biomarker calprotectin, in both blood and fecal samples was also assessed. For this purpose, five preterm infants received two daily doses (~109u2009CFU) of a 1u2009:u20091 mixture of Bifidobacterium breve PS12929 and Lactobacillus salivarius PS12934. Bacterial growth was detected by culture-dependent techniques in all the fecal samples. The phylum Firmicutes dominated in nearly all fecal samples while L. salivarius PS12934 was detected in all the infants at numerous sample collection points and B. breve PS12929 appeared in five fecal samples. Finally, a noticeable decrease in the fecal calprotectin levels was observed along time.

Early Gut Colonization of Preterm Infants: Effect of Enteral Feeding Tubes.

Objective: The aim of the study was to evaluate the potential colonization of nosocomial bacteria in enteral feeding systems and its effect on early gut colonization of preterm neonates. Methods: Mothers own milk, donor milk, and preterm formula samples obtained after passing through the external part of the enteral feeding tubes were cultured. In addition, meconium and fecal samples from 26 preterm infants collected at different time points until discharge were cultured. Random amplification polymorphism DNA and pulse field gel electrophoresis were performed to confirm the presence of specific bacterial strains in milk and infant fecal samples. Results: Approximately 4000 bacterial isolates were identified at the species level. The dominant species in both feces from preterm infants and milk samples were Staphylococcus epidermidis, S aureus, Enterococcus faecalis, E faecium, Serratia marcescens, Klebsiella pneumoniae, and Escherichia coli. All of them were present at high concentrations independently of the feeding mode. Random amplification polymorphism DNA and pulse field gel electrophoresis techniques showed that several bacteria strains were found in both type of samples. Furthermore, scanning electron microscopy revealed the presence of a dense bacterial biofilm in several parts of the feeding tubes and the tube connectors. Conclusions: There is a sharing of bacterial strains between the neonates’ gastrointestinal microbiota and the feeding tubes used to feed them.

Perinatal Microbiomes Influence on Preterm Birth and Preterms' Health: Influencing Factors and Modulation Strategies.

ABSTRACT Microbial communities inhabiting the human host play important roles in maintaining health status, including reproduction and early life programming, which is particularly important in the context of preterm neonates’ health. Preterm birth (PTB) is often the result of a microbial dysbiosis or infection. In addition, preterm neonates experience different levels of organ immaturity and an abnormal gut microbiota establishment, as compared to full-term neonates. This exacerbates their developmental problems and can have negative consequences at systemic level. In addition, preterm babies are commonly exposed to delayed enteral feeding and hospital environments, which increases the risk of short- and long-term health problems. Some of these clinical conditions, such as necrotizing enterocolitis or sepsis, may be life threatening, whereas others may translate into life-long conditions, including cognitive problems. Increasing scientific interest has focused on understanding developmental problems in preterm neonates related to abnormalities in the settlement of their microbial communities, with the final goal of selecting appropriate microbiome-targeted strategies (eg, probiotics), to reduce preterm health risks and improve overall quality of life. This review aims to summarize current knowledge on microbiological factors influencing PTB initiation and gastrointestinal development, and on the health consequences to the preterm neonate. Scientific evidences on dietary strategies reducing PTB incidence and minimizing sequelae in this particularly sensitive human group subpopulation are also discussed.Microbial communities inhabiting the human host play important roles in maintaining health status, including reproduction and early life programming, which is particularly important in the context of preterm neonates’ health. Preterm birth (PTB) is often the result of a microbial dysbiosis or infection. In addition, preterm neonates experience different levels of organ immaturity and an abnormal gut microbiota establishment, as compared to full-term neonates. This exacerbates their developmental problems and can have negative consequences at systemic level. In addition, preterm babies are commonly exposed to delayed enteral feeding and hospital environments, which increases the risk of short- and long-term health problems. Some of these clinical conditions, such as necrotizing enterocolitis or sepsis, may be life threatening, whereas others may translate into life-long conditions, including cognitive problems. Increasing scientific interest has focused on understanding developmental problems in preterm neonates related to abnormalities in the settlement of their microbial communities, with the final goal of selecting appropriate microbiome-targeted strategies (eg, probiotics), to reduce preterm health risks and improve overall quality of life. nThis review aims to summarize current knowledge on microbiological factors influencing PTB initiation and gastrointestinal development, and on the health consequences to the preterm neonate. Scientific evidences on dietary strategies reducing PTB incidence and minimizing sequelae in this particularly sensitive human group subpopulation are also discussed.

Bacterial Diversity of the Gastric Content of Preterm Infants during Their First Month of Life at the Hospital

Studies focused on the stomach microbiota are relatively scarce, and most of them are focused on the adult population. The aim of this work is to describe the bacterial communities inhabiting the gastric content (GC) of preterm neonates. For that purpose, GC samples were collected weekly from a total of 13 preterm neonates during their first month of life within their hospital stay. Samples were analyzed by using both culture-dependent and -independent techniques. The former allowed the isolation of bacteria belonging mainly to the genera Enterococcus, Staphylococcus, Streptococcus, Serratia, Klebsiella, and Escherichia. The cultured dominant species in the GC samples during all the hospitalization period were Enterococcus faecalis and Staphylococcus epidermidis. Multilocus sequence typing (MLST) analysis revealed the presence of high-risk clonal complexes associated with the hospital environment, which may colonize enteral feeding tubes. Similarly, the 16S rRNA sequencing showed that Streptococcus, Staphylococcus, Lactobacillus, Enterococcus, Corynebacterium, and Propionibacterium were the dominant genera present at 75% of the gastric samples. However, the genera Serratia, Klebsiella, and Streptococcus were the most abundant. Own mother’s milk (OMM) and donor milk (DM) were collected after their pass through the external feeding tubes to assess their bacterial content. OMM and DM had a similar bacterial pattern to GC. Based on these data, the GC of preterm neonates is dominated by Proteobacteria and Firmicutes and harbors high-risk bacterial clones, which may colonize enteral feeding tubes, and therefore the feeds that pass through them.

Bacteriological and Immunological Profiling of Meconium and Fecal Samples from Preterm Infants: A Two-Year Follow-Up Study

An abnormal colonization pattern of the preterm gut may affect immune maturation and exert a long-term influence on the intestinal bacterial composition and host health. However, follow-up studies assessing the evolution of the fecal microbiota of infants that were born preterm are very scarce. In this work, the bacterial compositions of fecal samples, obtained from sixteen 2-year-old infants were evaluated using a phylogenetic microarray; subsequently, the results were compared with those obtained in a previous study from samples of meconium and feces collected from the same infants while they stayed in the neonatal intensive care unit (NICU). In parallel, the concentration of a wide range of cytokines, chemokines, growth factors and immunoglobulins were determined in meconium and fecal samples. Globally, a higher bacterial diversity and a lower interindividual variability were observed in 2-year-olds’ feces, when compared to the samples obtained during their first days of life. Hospital-associated fecal bacteria, that were dominant during the NICU stay, seemed to be replaced, two years later, by genera, which are usually predominant in the healthy adult microbiome. The immune profile of the meconium and fecal samples differed, depending on the sampling time, showing different immune maturation statuses of the gut.

PO-0607c Administration Of Bifidobacterium Breve And Lactobacillus Salivarius, Two Strains Isolated From Human Milk, To Very Low And Extremely Low Birth Weight Preterm Infants:a Pilot Study

Preterm infant gut has been described as immature and colonised by an aberrant microbiota. Objectives Elucidate if administration of two probiotic strains isolated from human milk to preterm infants led to their presence in faeces. Secondarily, evolution of immunological compounds in blood and faecal samples was also assessed. Materials and methods Inclusion criteria: Birth weight <1,300 g, gestational age <29 weeks. Preterms received two daily doses (~109CFU) of a mixture of B. breve PS12929 and L. salivarius PS12934 after meconiorrhexis. Meconium samples were collected prior to and faecal and blood samples were collected weekly for up to 28 days. Faecal bacterial growth was detected by culture-dependent techniques. Cytokines, chemokines, growth factors and immunoglobulins were determined by multiplex technologies. The statistical analysis was performed using R2.15.3. Result and discussion Supplementation of five with this probiotics was effective in enhancing the levels of L. salivarius PS12934 that could be isolated from day 7 of intervention and its presence remained constant throughout the study; B. breve PS12929 was be detected later, after day 14, but had an increasing presence in the faecal samples. IL-4, IL-10 and IL-13 concentrations, related to anti-inflammatory processes, and IL-8 and MCP-1 were similar to those values previously reported for ‘late-preterms’ at 7 days of life, this may reflect the immumodulatory activity of the probiotic strains on this population. It demonstrated an increase in IgA since day 7. A reduction of calprotectin in faeces was observed throughout treatment. Globally, probiotic supplementation exerted a strong influence on gut colonisation.