Stool characteristics and tolerance measures are reported in Table 3. During all of the study periods, stool frequency was significantly higher in the BF group than in both formula groups, and there were no differences between formula groups. Stool consistency scores were significantly different among all of the groups, with the BF group being highest (range of means 4.0–4.1 ± 0.1), followed by PDX/GOS (3.3–3.5 ± 0.1) and control (2.9–3.1 ± 0.1). Means of parental-reported amount of gas were low (≤1.4, closest to “slight amount” of gas on the gassiness scale) in all of the groups throughout the study; however, it was significantly lower in the BF than in both formula groups in all of the study periods, with no differences between formula groups. Mean levels of fussiness were also low (≤1.2, closest to “slightly fussy” on the fussiness scale) during all of the study periods, and no significant differences were detected among groups.
There were no differences among study groups in the number of participants who experienced 1 or more adverse events. Also, there were no differences in the number of participants who experienced adverse events when analyzed by type of event, with 2 exceptions: differences were detected among groups in the number of participants with nasal/tear duct obstruction (control 2, PDX/GOS 1, BF 8; P = 0.045) and respiratory syncytial virus infection (control or PDX/GOS 0, BF 3; P = 0.028).
Quantification of Fecal Bacteria
FISH and qPCR were used to quantify fecal bifidobacteria and clostridia. Absolute counts of Bifidobacterium spp by FISH or qPCR are presented as box plots for comparison of results by these analytical methods (Fig. 2). By FISH, counts (log10 CFU/g stool) were significantly lower in the BF versus the control or PDX/GOS groups (P = 0.021 and P = 0.003, respectively) at baseline. No differences between groups were detected at 30 days. At 60 days, counts for the BF (median 9.86) were significantly higher than the counts for the control (9.36; P = 0.007) and PDX/GOS (9.58; P = 0.049) groups and were higher for the PDX/GOS versus the control group but did not reach statistical significance (P = 0.084). By qPCR, significant differences detected in Bifidobacterium spp among control, PDX/GOS, and BF groups demonstrated a time-dependent population shift from baseline to 30 and 60 days. At baseline, counts were significantly higher for the BF versus the control (P = 0.033) or PDX/GOS (P = 0.025) groups but not different between the control and PDX/GOS groups. At 30 days, a shift toward higher counts was observed for the PDX/GOS group. By 60 days, a pattern of higher counts for PDX/GOS was established: PDX/GOS counts, like BF counts, were significantly higher versus the control group (P = 0.002 and P = 0.001, respectively).
Across all of the fecal samples analyzed by qPCR, B longum was the most frequently detected species (71% of fecal samples from all of the study groups and time points), followed by B breve (33%), B animalis (28%), B bifidum (26%), B catenulatum (20%), B infantis (16%), and B adolescentis (4%). No group differences for B breve, B bifidum, or B adolescentis were detected at any measured time point (data not shown). Absolute counts of Bifidobacterium spp that demonstrated different time-dependent population shifts among the study groups are reported in Table 4. B longum closely paralleled the pattern demonstrated in total Bifidobacterium spp from baseline to 30 and 60 days and was similar to the BF group. No differences between groups were detected for counts of B animalis, B catenulatum, or B infantis at baseline or 60 days; however, at 30 days, counts of B animalis were similar between the BF and PDX/GOS but significantly higher in the BF versus the control group (P = 0.001), and B catenulatum was significantly higher in the PDX/GOS versus the BF group (P = 0.012). At 30 days, B infantis was significantly lower in the control versus the BF (P = 0.026) or PDX/GOS (P = 0.002) groups.
By FISH, absolute C lituseburense/C histolyticum counts (log10 CFU/g stool) at baseline, 30 days, and 60 days in the BF group (medians below detection limit at all time points) were significantly lower than the control (medians: 7.52, baseline; 7.13, 30 days; 7.34, 60 days; P < 0.001) and PDX/GOS (medians: 7.59, baseline; 7.16, 30 days; 7.40, 60 days; P < 0.001) groups. By qPCR, organisms in the C coccoides group were detected in 78% and C difficile in only 23% of the total fecal samples including the 3 study groups at all of the time points. Absolute counts of Clostridium spp are reported in Table 5. Counts of C coccoides group were lower in the BF versus both formula groups at all of the measured time points (P ≤ 0.002); formula groups were significantly different only at 60 days (P = 0.005). No differences were detected among groups for C difficile at baseline or between control and PDX/GOS groups at 30 or 60 days. Counts in the BF group were significantly lower versus PDX/GOS group at 30 days (P = 0.003) and versus control and PDX/GOS groups at 60 days (P = 0.013 and P = 0.003, respectively). Throughout the 60-day feeding period, however, few counts of C difficile or the C coccoides group reached a level >10 log10 CFU/g stool, and C difficile was below the detection limit by qPCR in >75% of infants from all of the groups at all of the time points.
Change From Baseline in Fecal Bacteria in Study Formula Groups
By FISH, no significant differences between formula groups in total Bifidobacterium spp or combined C lituseburense/C histolyticum as change from baseline to 30 or 60 days were detected (data not shown). Similarly, by qPCR, no significant differences between formula groups in change from baseline to 30 or 60 days were detected in total Bifidobacterium spp, B bifidum, B breve, B animalis, B adolescentis, C coccoides, or C difficile (data not shown). The change from baseline was significantly higher in PDX/GOS than in the control group for B catenulatum (percentage of infants with an increase: 27% vs 14%; P = 0.004) and B infantis (19% vs 2%; P = 0.024) at 30 days and for B longum (72% vs 59%; P = 0.035) at 60 days. Furthermore, a significantly larger change in the number of Bifidobacterium spp was detected in the PDX/GOS compared with the control group at both 30 (mean 0.82 ± 0.14 vs 0.31 ± 0.12; P = 0.008) and 60 days (mean 1.20 ± 0.19 vs 0.59 ± 0.18; P = 0.021) of feeding (Fig. 3).
Quantification of Fecal sIgA
The BF group showed significantly higher levels of fecal sIgA by ELISA (milligram per gram of stool) than control or PDX/GOS groups at baseline (medians 3.07, 0.48, 0.45; P < 0.001), 30 (medians 3.42, 0.86, 1.21; P < 0.001), and 60 days (medians 3.38, 1.09, 1.26; P < 0.001) of feeding. Levels of fecal sIgA at any time point or change of sIgA from baseline to 30 or 60 days of feeding were not significantly different between formula groups (data not shown).
The intestinal microbiota of BF infants is a model for early nutritional intervention, such as supplementation of infant formula with PDX and GOS. This supplementation may stimulate the microbiota at a stage in which immune maturation and consequent resistance to infections and allergic diseases can be influenced (7,14,20,21). Although levels of beneficial bifidobacteria tend to be higher in BF infants when compared with formula-fed infants (2,4,9), a bifidogenic effect can be achieved in infants receiving formula supplemented with prebiotics in amounts up to 10 g/day (13,15,16). We previously demonstrated that supplementation of PDX and GOS (4 g/L; 1:1 ratio) to routine cow's-milk–based formula is well tolerated, safe, and promotes normal growth (29,34). In the present study, infant formula added with this prebiotic blend promoted an increase in members of the Bifidobacterium genus (specifically, B infantis, B longum, and B catenulatum) in healthy term infants during a 60-day feeding period.
Bifidogenic effects for PDX and GOS in isolation have been demonstrated in adults and infants (25,35,36). In these studies, bifidobacteria were enumerated using traditional culture-based techniques, which can elicit lower counts than probe hybridization techniques (32). In particular, fecal anaerobes can be damaged if anaerobic incubation is not performed immediately after defecation (30,32). Bifidobacteria may also be overestimated in culture-dependent methods because total culturable counts are only a fraction of the total intestinal microbiota (32), whereas molecular methods more accurately quantify bacterial populations (37).
In the present study, 2 quantitative molecular culture-independent techniques, FISH and qPCR, were used to analyze potential shifts in the infant fecal microbiota. These methods enable detection of less abundant organisms and allow species-level discrimination (37). Although no difference in change from baseline of total bifidobacteria between groups was observed in the present study, significantly higher changes from baseline of B longum and B infantis were observed in the PDX/GOS compared with the control group. Along with B breve, B longum and B infantis are the most common Bifidobacterium spp found in infants and they predominate in the intestinal microbiota of BF infants (38). Higher absolute counts of total bifidobacteria and B longum in the PDX/GOS compared with the control group observed at 30 days became statistically significant by 60 days of feeding. Furthermore, the control group but not the PDX/GOS group had significantly lower counts of total bifidobacteria, B longum, B infantis, and B animalis than the BF group, corroborating that the prebiotic supplementation may have modified the gut microbiota toward a profile observed in BF infants.
Subtle differences in the microbiota of BF infants and formula-fed infants may occur at the species level. Prebiotic intervention has been linked to a shift in the microbiota of formula-fed infants toward a greater number of Bifidobacterium spp, making it closer to that of BF infants (17). A higher number of bifidobacterial species may be necessary for postnatal maturation of a normal immune system, which requires constant microbial stimulation by the developing intestinal microbiota (1,8). In the present study, the PDX/GOS group developed a higher number of Bifidobacterium spp compared with the control group. We also observed a time-dependent shift toward higher counts of total bifidobacteria and B longum during the 60-day feeding period, possibly reflecting maturation of the gut microbiota. A similar shift in infants fed infant formula supplemented with GOS/FOS has been described by others (39).
Although some previous studies have reported a potentially beneficial prebiotic effect of lowering pathogenic species, including C difficile(16,19), this effect was not observed in other studies (9,13,40), including our study. Bifidobacteria are among the initial and most prevalent colonizers of the gastrointestinal tract. In contrast, clostridia, including members of the C coccoides group, appear later in the colonization process, as demonstrated by their relatively low levels in infants (6,41). In vitro experiments suggest that the suppression of C difficile by some prebiotics is not linked to a competition with bifidobacteria, but likely involves multiple barrier factors that inhibit pathogen growth (42). In the present study, clostridia appeared at a much lower level than bifidobacteria, yet increased in all of the 3 groups during the 60-day study period, possibly reflecting normal maturation toward higher, typical adult-like numbers (41).
In agreement with several previous reports (13,14,29,34,36), we demonstrated a stool-softening effect with PDX/GOS, which was closer to that of the BF than the control group. This effect is potentially beneficial to help manage the hard stools or constipation that may affect formula-fed infants (43). Transition from breast to bottle has been shown to occur during the 3 months preceding onset of constipation in nearly 25% of constipated children in the first 2 years of life (44). Harder stools, typical of formula-fed infants, have less water-soluble and complex carbohydrates when compared with stools of BF infants (43). The protective effect of fiber against constipation is the result of the osmotic stimulation caused by the short-chain fatty acids generated during fermentation of fiber by colonic bacteria, in addition to the water-holding capacity of undigested components of fiber (45).
Infant growth in the present study was similar among groups, with the exception of a lower weight growth rate at 60 but not 30 days in the BF group. No differences in the incidence of adverse events were observed between the prebiotic-supplemented and the unsupplemented formula groups, and the only significantly higher incidence of adverse events occurred in the BF group (nasal/tear duct obstruction and respiratory syncytial virus infection).
One potential concern linked to supplementation of infant formula with prebiotics is excessive gas formation. The addition of PDX/GOS to infant formula had no negative effect on gassiness, which is consistent with previous studies (29,34). This indicates that the amount of prebiotics in the supplemented formula, 4 g/L, achieves a bifidogenic effect and at the same time promotes a health benefit of stool softening without excessive gas production. The induction of more watery stools is recognized by the European Society for Gastroenterology, Hepatology, and Nutrition as a relevant benefit of prebiotics in infants experiencing constipation (46). Collectively, the stimulation of beneficial bacteria and the benefit of softer stools indicate that the PDX/GOS blend indeed meets the European Society for Gastroenterology, Hepatology, and Nutrition's and the Food and Agriculture Organization of the United Nations's definition of a prebiotic (ie, it confers a health benefit on the host associated with modulation of the microbiota) (46,47).
Formula-fed infants who do not benefit from the protective sIgA transferred from breast milk may benefit from measures aimed to increase production of endogenous sIgA. Increased sIgA has been associated with the feeding of PDX to an animal model (28) and GOS in combination with FOS to infants (18,19). In the present study, the BF group had consistently higher sIgA than the formula groups, but no statistically significant differences in sIgA were observed between formula groups. In another study, although the addition of prebiotics to infant formula elicited no increase in total sIgA, a positive correlation between anti-poliovirus sIgA and B longum/B infantis species after vaccination was observed (48). Therefore, such a potential effect of infant formula with PDX/GOS on sIgA warrants further evaluation.
Not only the type and amount of prebiotics but also the analytical methods used to identify and quantify bacteria can influence fecal microbiota results (9,32,49). The 2 molecular techniques chosen in our study corroborated our microbiota results with complementary analyses. The high detection limit of FISH (which does not allow quantification of extremely low bacterial numbers in fecal samples) (37) may have been responsible for the absence of statistically significant differences between formula groups that were detected by qPCR. Differently from conventional PCR, qPCR monitors complete DNA amplification at all of the stages, not just the plateau phase, and consequently detects decreasing differences in PCR product abundance, which is appropriate for accurate bacteria quantification (6). In addition, qPCR provides high specificity by using specific primers and probes (17,49).
When the criterion standard of infant nutrition, breast milk, is not available, supplementation of infant formula with prebiotics early in life may offer an opportunity to affect microbiota development (34). The results of the present study demonstrate that early nutritional intervention of infant formula with 4 g/L of a PDX/GOS 1:1 blend has a bifidogenic effect and promotes softer stools that are closer to those of BF infants than infants fed an unsupplemented formula. As such, the PDX/GOS–supplemented formula may be helpful to manage or prevent early constipation in formula-fed infants.
We thank study investigators and their staff for their cooperation. The participation of parents and infants in the present study is gratefully acknowledged. Additionally, we thank Jennifer L. Wampler, PhD, for contributions to manuscript development, and Laura B. Beck, Rebecca A. Hirsch, and Bryan Liu, MD, for management of study sites.
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Keywords:Copyright 2012 by ESPGHAN and NASPGHAN
bifidogenic effect; galacto-oligosaccharides; infants; polydextrose; prebiotics