Silvia Fanaro

Intestinal microflora in early infancy: composition and development.

The neonatal intestinal microbiota is a complex ecosystem composed of numerous genera, species and strains of bacteria. This enormous cell mass performs a variety of unique activities that affect both the colonic and systemic physiology. Its primary activities include nutritive, metabolic, immunological and protective functions. Most studies of infants have been based on faecal samples using the classical plating techniques with culturing on specific media. The limitations of these methods must be taken into account when evaluating the varying results of the different studies. The establishment of the gut microbial population is not strictly a succession in the ecological sense; it is rather a complex process influenced by microbial and host interactions and by external and internal factors. The climax intestinal flora is attained in successive stages. The foetal intestine is sterile and bathed in swallowed amniotic fluid. Following delivery, multiple different antigens challenge the intestine of the newborn. The maternal intestinal flora is a source of bacteria for the neonatal gut. The bacterial flora is usually heterogeneous during the first few days of life, independently of feeding habits. After the first week of life, a stable bacterial flora is usually established. In full‐term infants a diet of breast milk induces the development of a flora rich in Bifidobacterium spp. Other obligate anaerobes, such as Clostridium spp. and Bacteroides spp., are more rarely isolated and also enterobacteria and enterococci are relatively few. During the corresponding period, formula‐fed babies are often colonized by other anaerobes in addition to bifidobacteria and by facultatively anaerobic bacteria; the development of a “bifidus flora” is unusual. In other studies the presence of a consistent number of bifidobacteria in infants delivered in large urban hospitals has not been demonstrated, whether the babies were bottle fed or exclusively breastfed. The predominant faecal bacteria were coliforms and bacteroides. According to these studies, environmental factors may be more important than breastfeeding in gut colonization after delivery. Environmental factors are indeed extremely important for the intestinal colonization of infants born by caesarean section. In these infants, the establishment of a stable flora characterized by a low incidence of Bacteroides spp. and by the isolation of few other bacteria is consistently delayed. In extremely low‐birthweight infants, hospitalization in neonatal intensive care units, characterized by prolonged antibiotic therapy, parenteral nutrition, delayed oral feedings and intubation seems to affect the composition of the intestinal microbiota. The gut is colonized by a small number of bacterial species; Lactobacillus and Bifidobacteria spp. are seldom, if ever, identified. According to the few studies so far performed, the predominant species are Enterococcus faecalis, E. coli, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus epidermidis and Staphylococcus haemolyticus. Hygienic conditions and antimicrobial procedures strongly influence the intestinal colonization pattern.

Dosage-related bifidogenic effects of galacto- and fructooligosaccharides in formula-fed term infants.

Background Human milk oligosaccharides have been shown to stimulate selectively the growth of Bifidobacteria and Lactobacilli in the intestine. In this study, the bifidogenic effect of an experimental prebiotic oligosaccharide mixture consisting of low-molecular-weight galactooligosaccharides and high-molecular-weight fructooligosaccharides was analyzed in 90 term infants. Methods Two test formulas were supplemented with either 0.4 g/dL or with 0.8 g/dL oligosaccharides. In the control formula, maltodextrin was used as placebo. At study day 1 and study day 28, the fecal species, colony forming units (cfu) and pH were measured and stool characteristics, growth, and side effects were recorded. Results At study day 1, the median number of Bifidobacteria did not differ among the groups (0.4 g/dL group, mean [interquartile range] 8.5 [1.9] cfu/g; 0.8 g/dL group, 7.7 [6.1] cfu/g; and the placebo group, 8.8 [6.1] cfu/g) (figures in square brackets are interquartile range). At the end of the 28-day feeding period, the number of Bifidobacteria was significantly increased for both groups receiving supplemented formulas (the 0.4 g/dL group, 9.3 [4.9] cfu/g; the 0.8 g/dL group, 9.7 [0.8] cfu/g) versus the placebo group (7.2 [4.9] cfu/g, P < 0.001). This effect was dose dependent (0.4 g/dL versus 0.8 g/dL, P < 0.01). The number of Lactobacilli also increased significantly in both groups fed the supplemented formulas (versus placebo, P < 0.001), but there was no statistically significant difference between the group fed formula with 0.4 g/dL oligosaccharides and the group fed formula with 0.8 g/dL oligosaccharides. The dosage of supplement significantly influenced the change in fecal pH (P < 0.05) (placebo, pH 5.5–6.1; 0.4 g/dL formula, pH 5.48–5.44; 0.8 g/dL formula, pH 5.54–5.19). Slight changes in the stool frequency resulted in a significant difference between the placebo group and the group fed the 0.8 g/dL formula at day 28 (P < 0.01). Supplementation had a significant dose-dependent influence on stool consistency (0.8 g/dL versus placebo, P < 0.0001; 0.8 g/dL versus 0.4 g/dL, P < 0.01). Supplementation had no influence on the incidence of side effects (crying, regurgitation, vomiting) or growth. Conclusions These data indicate that supplementation of a term infants formula with a mixture of galacto- and fructooligosaccharides has a dose-dependent stimulating effect on the growth of Bifidobacteria and Lactobacilli in the intestine and results in softer stool with increasing dosage of supplementation.

Galacto-oligosaccharides and long-chain fructo-oligosaccharides as prebiotics in infant formulas: A review

The present review summarizes clinical and experimental data concerning the possible effects of a prebiotic mixture of short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides. The results from several studies, made up of over 400 preterm and term infants, clearly demonstrate that the prebiotic mixture under examination specifically stimulates the growth of bifidobacteria and lactobacilli and reduces the growth of pathogens. As a consequence of the changed intestinal flora by the dietary galacto-oligosaccharides and fructo-oligosaccharides, the faecal pH values and the short-chain fatty acid pattern were similar to those found in breastfed infants. In addition, the stool consistency was the same as in breastfed infants. In vitro experiments have demonstrated that the specific short-chain fatty acid pattern, at a pH similar to that found in faecal samples of breastfed infants, reduces the growth of pathogens in a dose-dependent manner but does not influence the growth of bifidobacteria and lactobacilli. In an animal vaccination model, the prebiotic mixture improved the response to vaccination. In an allergy model (sensitization by ovalbumin), the allergic reaction was reduced by the prebiotic mixture. The data obtained from animal experiments are in agreement with preliminary data from clinical trials which indicate a reduced allergic response (reduced plasma IgE/IgG4 ratio) and reduced episodes of upper airway infection during the first year of life. Conclusion: Experimental evidence demonstrates that the prebiotic mixture employed in these studies modulates the intestinal flora and modulates the immune system as human milk does. There are sufficient experimental data to put forward the hypothesis that substances like the prebiotic mixture under study will substantially contribute to the improvement of the protective properties of infant formulas.

Acidic oligosaccharides from pectin hydrolysate as new component for infant formulae : Effect on intestinal flora, stool characteristics, and pH

Objectives: To come even closer to the functional composition of human milk, acidic oligosaccharides (AOS) from pectin were added to well known neutral prebiotics (galacto-oligosaccharides (GOS) and long-chain fructo-oligosaccharides (FOS)). The effect of AOS and GOS/FOS/AOS on intestinal flora, stool characteristics as well as acceptance and tolerance was investigated. Methods: Human milk contains 75% to 85% neutral and 15% to 25% acidic oligosaccharides. In this prospective, randomized, double blind study, a mixture of 80% neutral oligosaccharides (from long-chain galacto- and long-chain fructo-oligosaccharides) with 20% acidic oligosaccharides derived from pectin hydrolysis was investigated. Forty-six term infants were fed a standard formula supplemented with either maltodextrin as control (n = 15), or with 0.2 g acidic oligosaccharides (n = 16), or with the latter plus 0.6 g neutral oligosaccharides (mixture of galacto- and fructo-oligosaccharides; n = 15). Fecal flora using plating technique and pH were measured. Stool characteristics and possible side effects (crying, vomiting, and regurgitation) were recorded. Results: There was no difference in the bifidobacteria counts between the control and the group supplemented with acidic oligosaccharides alone (8.75 ± 0.50 vs. 8.58 ± 0.94 log colony forming units [CFU]/g stool). In infants fed the combination of acidic and neutral oligosaccharides, bifidobacteria were increased (9.61 ± 0.70 log CFU/g stool; P < 0.01). The same pattern was observed with lactobacilli. Stool consistency was softest in infants fed the complete oligosaccharide mixture, but also in those fed formula supplemented with acidic oligosaccharides alone, the stool consistency was significantly softer compared with the control group. Fecal pH increased in the controls, remained constant in acidic oligosaccharides alone, and decreased in the complete mixture of oligosaccharides group. Conclusion: There was no difference in growth, crying, vomiting, and regurgitation patterns between the groups. In summary, acidic oligosaccharides from pectin hydrolysate are well tolerated as ingredient in infant formulae but do not affect intestinal microecology.

Prebiotic concept for infant nutrition.

In the neonatal period, the intestine is colonised in a stepwise process that depends on mode of delivery, environmental factors, bacterial interactions, and the host itself resulting in a colonisation with a complex heterogeneous bacterial flora. Oligosaccharides have been identified as an important prebiotic factor of human milk As long as analogues of human milk oligosaccharides are not available now and in the near future it is aimed to resemble the prebiotic effect of human milk by oligosaccharides from available sources. In the present study in preterm infants, a mixture of 90% galacto‐oligosaccharides and 10% fructo‐oligosaccharides has been tested. The mixture of GOS/FOS was composed to mimic the molecule size distribution of human milk oligosaccharides. Microbiological analysis of the faces was performed before and 7, 14, and 28 days after start of supplementation and stool characteristics have been recorded. Maltodextrin was used as placebo and infants fed human milk have been used as reference. After a 28 days feeding period, the number of bifidobacteria of the group fed the oligosaccharide supplemented formula was in the upper range of the reference group whereas the numbers of the group fed the formula supplemented with the placebo were in the lower range of the reference group (placebo: 7.9 ± 0.83 and GOS/FOS mixture: 10,0 ± 2.05 log 10 CFU/g wet stool; reference (M ± SD): 7.14‐10.7 log 10 CFU/g wet stool). Stool characteristics in the group fed the supplemented formula were close to those found in the human milk fed infants. In summary, supplementation of a preterm formula with a mixture of galacto‐ and fructo‐oligosaccharides has a stimulating effect on the growth of bifidobacteria in the intestine and results in more frequent produced and softer stools. Thus, prebiotic mixtures such like the studied oligosaccharide mixture might help in improving intestinal tolerance to enteral feeding in preterm infants.

Effects of a new mixture of prebiotics on faecal flora and stools in term infants.

A double‐blind, randomized, controlled study was performed in 90 full term infants to evaluate dose‐related bifidogenic effects of a new synergistic mixture of galacto‐oligosaccharides (GOS) and fructo‐oligosacharides (FOS). The GOS/FOS mixture showed a dose‐dependent stimulatory effect on the intestinal growth of bifidobacteria. Also stool consistency and faecal pH were positively affected.

Galacto-oligosaccharides are bifidogenic and safe at weaning: a double-blind randomized multicenter study.

Objectives: The primary objective of this study was to determine the bifidogenic effect of galacto-oligosaccharides (GOS) in a follow-on formula and the effects on other intestinal bacteria. Secondary objectives were the effects on stool characteristics, growth, and general well-being. Participants and Methods: In a multicenter, double-blind study, 159 healthy infants, formula-fed at enrollment (at 4–6 months), were randomized to an experimental follow-on formula supplemented with 5 g/L (GOS) (77 infants), or to a standard follow-on formula (control, 82 infants). Infants were evaluated at enrollment (study day 1 = sd1), after 6 weeks (study day 2 = sd2), and after an additional 12 weeks (study day 3 = sd3). At each study day, a fresh stool sample for the bacterial counts was collected, and the growth parameters were measured. At sd2, urinary specimens were collected for the evaluation of urinary osmolarity. Results: At sd2 and sd3, the GOS group had a higher median number (colony-forming units per gram of stool) of bifidobacteria than did the control group (sd2 GOS 9.2 × 109 vs control 4.4 × 109, P = 0.012); (sd3 GOS 7.2 × 109 vs control 2.4 × 109, P = 0.027). Other bacteria did not show any significant differences between the 2 groups at all study days. The GOS produced softer stools but had no effect on stool frequency. The urinary osmolarity (mOsm/L) at sd2 was comparable in both groups. Supplementation had no influence on the incidence of gastrointestinal side effects or on the growth of the infants. Conclusions: These data indicate that the addition of GOS (5 g/L) to a follow-on formula positively influences the bifidobacteria flora and the stool consistency in infants during the supplementation period at weaning. No local or systemic side effects were recorded.

Minor feeding problems during the first months of life: effect of a partially hydrolysed milk formula containing fructo- and galacto-oligosaccharides.

Background: Colic, regurgitation and constipation are common feeding problems in formula‐fed infants that might benefit from dietary treatment. A formula containing fructo‐ and galacto‐oligosaccharides, partially hydrolysed proteins, low levels of lactose and palmitic acid in the β position and higher density has been tested to reduce the occurrence of these symptoms. The aim of this prospective study was to describe the effects of such a formula in infants with minor gastrointestinal disorders. Methods: An observational prospective trial involving practising Italian paediatricians was performed. Formula fed‐infants up to 90 d of age with minor gastrointestinal problems such as infantile colics and/or regurgitation and/or constipation were enrolled in the study from January 2001 to May 2001. The study was completed within 14 d of treatment. On days 1, 7 and 14 the infants were visited by the paediatricians. Parents were given a structured diary to record daily episodes of colic, regurgitation and type and number of stools. Results: Of the 932 infants enrolled, 604 completed the study. Of the 214 infants with colic, 169 (79%) demonstrated a reduction in frequency of colic from 4.1 ± 2.0 per day at the beginning of the study to 2.0 ± 1.8 at the end of the study (I.C. 95%: 1.72–2.39; p < 0.005). A reduction in the number of episodes of colic of 1.8 per day at the beginning of the study (I.C. 95%: 1.49–2.11; p < 0.05) was recorded between day 1 and day 7, and of 0.26 (I.C. 95%: 0.15–0.37; p < 0.05) between day 7 and day 14. Of the 201 infants with regurgitation problems, 141 (70%) demonstrated a reduction of frequency of the symptoms from 4.2 ± 2.0 per day at the beginningof the study to 2.1 ± 2.2 at the end of the study (I.C. 95%: 1.75–2.35; p < 0.005). A reduction of 1.87 in the number of regurgitation episodes was reported between day 1 and day 7 (I.C. 95%: 1.57–2.16; p < 0.05) and of 0.18 (I.C. 95%: 0.06–0.31; p < 0.05) between day 7 and day 14. Of the 232 infants with constipation, 147 (63%) demonstrated an increase in the daily number of stools of 0.42 (I.C. 95%: 0.5–0.3; p < 0.005). An increase in stool frequency of 0.41 (I.C. 95%: 0.51–0.23; p < 0.05) was reported between day 1 and day 7, and of 0.04 (I.C. 95%: 0.22–0.14; p= ns) between day 7 and day 14. Parents’evaluation of the formula was 7.9 ± 1.8 (score 0–10); 550 parents (91%) gave a positive judgement (score >6). The evaluation by the paediatricians of the improvement in symptoms after the treatment was 8.2 ± 1.5; 574 (95%) a positive effect (score >6).

Neonatal Onset Intestinal Failure: An Italian Multicenter Study

OBJECTIVE To describe the natural course of intestinal failure with onset in the neonatal period to provide data regarding the occurrence and to provide a population-based survey regarding the spectrum of underlying diseases. STUDY DESIGN We performed a retrospective chart review including infants admitted to the neonatal intensive care unit of 7 Italian tertiary care centers. Intestinal failure was defined as a primary intestinal disease that induces the need of total parenteral nutrition (PN) for more than 4 weeks or the need of partial PN for more than 3 months. RESULTS The total number of live births during the study time within the enrolled institutions was 30 353, and the number of newborns admitted to the neonatal intensive care unit was 5088. Twenty-six patients satisfied the definition of intestinal failure; thus the occurrence rate of intestinal failure was 0.1% among live-birth newborns and 0.5% among infants at high risk. The main underlying diseases leading to intestinal failure in neonatal age were congenital intestinal defects (42.3%), necrotizing enterocolitis (30.8%), severe intestinal motility disorder (11.5%), intestinal obstruction (7.7%), structural enterocyte defects (3.8%), and meconium peritonitis (3.8%). After a follow-up of 36 months, 84.6% of patients achieved intestinal competence, 1 patient was still receiving home PN, 1 patient underwent transplantation, and 2 patients died. Cholestatic liver disease was diagnosed in 54% of observed children. CONCLUSION An understanding of the incidence, causes, and natural history of intestinal failure would be helpful to appropriately allocate resources and to plan clinical trials.

Advances in the modulation of the microbial ecology of the gut in early infancy.

It is now generally accepted that the microbiota of the human gut may influence health and well‐being. Lactic acid bacteria are the most important microorganisms associated with these beneficial effects and the elevated bifidobacterial count may be one of the greatest advantages that breastfed infants have over infants fed with milk formulas. Several studies relative to the selective growth stimulation of bifidobacteria, both in vitro and in vivo, are reported in this review. Over the years, diverse human milk components have been identified as the specific factors able to modulate the growth of bifidobacteria. Even if there is a certain agreement that the bifidogenic activity of human milk may be based not on single growth substances, but on a complex set of interacting factors, the present state of knowledge indicates that the use of non‐digestible but fermentable carbohydrates may be an easy and reliable method to influence the growth of lactic acid bacteria. In this context, some of the characteristics of the major physiological effects of inulin‐type fructans, of galacto‐oligosaccharides, but also of lactoferrin, a milk whey protein fraction with purported bifidogenic activity, are briefly examined.