AJCN EB Program 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chouraqui, J. P.
Right arrow Articles by Steenhout, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chouraqui, J. P.
Right arrow Articles by Steenhout, P.
Agricola
Right arrow Articles by Chouraqui, J. P.
Right arrow Articles by Steenhout, P.
American Journal of Clinical Nutrition, Vol. 87, No. 5, 1365-1373, May 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Assessment of the safety, tolerance, and protective effect against diarrhea of infant formulas containing mixtures of probiotics or probiotics and prebiotics in a randomized controlled trial1,2,3,4

Jean Pierre Chouraqui, Dominik Grathwohl, Jean Marc Labaune, Jean Michel Hascoet, Ines de Montgolfier, Michèle Leclaire, Mariana Giarre and Philippe Steenhout

1 From the Gastroenterology and Nutrition Unit, Department of Pediatrics, Centre Hospitalier Universitaire de Grenoble, Grenoble, France (JPC); the Nestlé Research Center, Lausanne, Switzerland (DG, MG); the Hôpital de la Croix Rousse, Lyon, France (JML); the Service de Néonatologie; Maternite Régionale, Nancy University, Nancy, France (JMH); the Hôpital d'Enfants Armand Trousseau, Paris, France (IdM); the Service de Néonatologie, Hôpital de La Conception, Assistance Publique, Hôpitaux de Marseille, Marseille, France (ML); and Nestlé Nutrition, Vevey, Switzerland (PS)

2 Mariana Giarre is deceased.

3 Supported by Nestlé Nutrition, Vevey Switzerland.

4 Address reprint requests and correspondence to JP Chouraqui, Pediatric Gastroenterology and Nutrition, CHU de Grenoble, BP 217-38043, Grenoble Cedex 07, France. E-mail: jpchouraqui{at}chu-grenoble.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Probiotics and prebiotics are considered to be beneficial to the gastrointestinal health of infants.

Objective: The objective was to evaluate infant formulas containing probiotics and synbiotics (combinations of probiotics and prebiotics) for safety and tolerance.

Design: In a prospective, controlled, double-blind, randomized trial, healthy full-term infants were exclusively fed a control formula or study formulas containing Bifidobacterium longum BL999 (BL999) + Lactobacillus rhamnosus LPR (LPR), BL999 + LPR + 4 g/L of 90% galactooligosaccharide/10% short-chain fructooligosaccharide (GOS/SCFOS), or BL999 + Lactobacillus paracasei ST11 (ST11) + 4 g/L GOS/SCFOS from ≤2 to 16 wk of age (treatment period). Safety and tolerance were assessed based on weight gain during the treatment period (primary outcome) as well as recumbent length, head circumference, digestive tolerance, and adverse events (secondary outcomes), which were evaluated at 2, 4, 8, 12, 16, and 52 wk of age.

Results: Two hundred eighty-four infants were enrolled. During the treatment period, difference in mean weight gain between control and study formula groups in both the intention-to-treat and per-protocol populations were within the predefined equivalence boundaries of ±3.9 g/d, indicating equivalent weight gain. Secondary outcomes did not show significant differences between groups during the treatment period.

Conclusion: Infants fed formulas containing probiotics or synbiotics show a similar rate in weight gain compared with those fed a control formula and tolerate these formulas well.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Breastfed infants are generally healthier than formula-fed infants, especially with respect to their ability to fend off infections (1). Some of the health benefits of human milk have been attributed partly to factors that modulate the development of a normal gut microbiota (1). These factors, which include complex oligosaccharides, are thought to selectively stimulate the growth of bacteria considered to be beneficial, such as bifidobacteria and lactobacilli (2, 3), and inhibit the growth of potentially pathogenic bacteria (4, 5).

Hence, the development of improved infant formulas has focused on emulating the beneficial effects of breast milk by, among other approaches, supplementing formulas with specific probiotics or oligosaccharides (prebiotics) that selectively stimulate the growth or metabolic activity of potentially beneficial indigenous bacteria such as bifidobacteria. A number of clinical studies in which the formula of infants was supplemented with probiotics suggest that some probiotics may indeed have beneficial effects in managing and preventing gastrointestinal (GI) infections and diarrhea (6-10), prevent the onset of allergy, and be useful in the treatment of atopic disease (11, 12). The potential benefits of prebiotics are multifaceted; in addition to enhancing growth of indigenous bacteria, prebiotics are fermented in the large intestine, yielding short-chain fatty acids (SCFAs). These create an acidic environment that inhibits growth of potentially pathogenic bacteria (13) and also serve as an energy source for the gut epithelium (14). Furthermore, it has also been suggested that SCFAs aid the absorption of magnesium, calcium, and iron (15, 16).

The combination of probiotics and prebiotics (synbiotics) has been proposed to have a synergistic effect by both ensuring survival of delivered probiotics and stimulating the growth of selected indigenous bacteria (13, 17). However, clinical studies showing the effects of feeding synbiotics to infants are scarce. A recent study (18) has shown that a standard infant formula supplemented with a mixture of a probiotic (Bifidobacterium longum BL999) with the prebiotics galactooligosaccharide (GOS) and short-chain fructooligosaccharide (SCFOS) is safe and well tolerated by healthy infants. In the current study, we aimed to systematically evaluate 3 formulas containing different mixtures of the probiotics B. longum BL999, Lactobacillus rhamnosus LPR, and Lactobacillus paracasei ST11 as well as a mixture of probiotics and the prebiotics GOS and SCFOS for their safety and effect on the incidence of diarrhea in infants. The benefit of L. rhamnosus LPR in the prevention and treatment of diarrhea is well documented in clinical studies (19) and has been reviewed (9, 20, 21). B. longum BL999 is a human GI tract isolate that is currently consumed as part of some milk-based products. It has been used safely in clinical studies in both infants and adults and has been shown to transiently colonize the gut of infants after antibiotic treatment without causing any side effects (22-24). Finally, L. paracasei ST11, an isolate from a healthy infant, has also been safely used in infants and has been shown to be useful in ameliorating nonrotavirus-induced diarrhea (25, 26).


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Subjects were healthy infants recruited from mothers who had chosen not to breastfeed and had decided to feed their infants exclusively with formula from the time of enrollment until infants were at least 16 wk old. To be included in the study, infants had to be healthy, full-term (gestational age between 37 and 42 wk), ≤14 d old, singletons, and weigh between 2500 and 4500 g. Infants were excluded from the study if they had major deformities or cardiovascular, GI, renal, neurological, or metabolic illnesses; had required hospital admission for intensive care or for ≥3 d; were born to mothers with diabetes; or if their parents were expected to have difficulty complying with the feeding regimen.

This study was conducted in accordance with the principles and rules of the Declaration of Helsinki and adhered to Good Clinical Practice guidelines of the International Conference on Harmonization. It was approved by the Ethics Committee of Grenoble (7 April 2004), and written informed consents were signed by parents or legal guardians before enrollment.

Trial design
The current study was a prospective, double-blind, reference-controlled, parallel-group, randomized trial, conducted in 5 centers in France: CHU of Grenoble; Hôpital de la Croix Rousse, Lyon; Service de Néonatologie, Maternité Régionale, Nancy; Hôpital d'Enfants Armand Trousseau, Paris; and Service de Néonatologie, Hôpital de La Conception, Marseille. The total treatment period lasted {approx}8 mo (15 October 2004 to 21 June 2005), and each infant was treated for 14–16 wk.

On enrollment, infants were assigned into 1 of 4 groups by block randomization using sex and center as strata. Formulas were coded by the sponsor, and both the investigators and the infants’ parents were blinded to the formulas. Each group received either a control formula lacking any probiotics or prebiotics, or one of 3 study formulas containing different mixtures of probiotics or synbiotics: B. longum BL999 and L. rhamnosus LPR (group BL999 + LPR); B. longum BL999, L. rhamnosus LPR, and GOS/SCFOS, (group BL999 + LPR + GOS/SCFOS); or B. longum BL999, L. paracasei ST11, and GOS/SCFOS (group BL999 + ST11 + GOS/SCFOS). Baseline measurements were taken at enrollment, and parents received the corresponding formulas for their infants, along with instructions on preparation and feeding. During the treatment period (0–4 mo) infants were fed ad libitum exclusively with their allocated formulas starting on the day of enrollment (1–14 d of age) through 112 d (16 wk) of age. Follow-up visits took place when infants were 11–17, 28–42, 49–63, 77–91, 105–119, and {approx}365 d old (2, 4, 8, 12, 16 and 52 wk, respectively). The observational period consisted of data from 16 to 52 wk (4 to12 mo).

Parents kept daily records of the infants’ intake of formula, supplementary foods, and any medication or treatments. Three days before and following a visit, parents also recorded stool characteristics, frequency of flatulence, and behavior of infants. At each visit investigators took anthropomorphic measurements; reviewed compliance (based on diet records kept by parents and by comparing the number of cans of formula issued with the number remaining at each visit), intake, and infants’ tolerance (based on records kept by the parents); and assessed any incidents of morbidity.

Formulas and bacterial strains
All of the formulas were powdered starter formulas intended for full nutritional support of infants from birth to 6 mo and contained the same amounts of protein, carbohydrate, fat, vitamins, minerals, and long-chain polyunsaturated fatty acids. The control formula was unsupplemented formula (Nan; Nestec SA, Konolfingen, Switzerland), whereas the study formulas were control formula supplemented with mixtures of probiotic bacteria strains or probiotics and prebiotics (Table 1Go). The probiotic strains were B. longum BL999 (ATCC: BAA-999 designation BB536, Morinaga, Japan), L. rhamnosus LPR (CGMCC 1.3724), and L. paracasei ST11 (CNCM I-2116), with the number of colony-forming units in each formula shown in Table 1Go. The prebiotic mixture (0.4 g/100 mL) contained 90% GOS and 10% SCFOS. All formulas were manufactured according to Good Manufacturing Practices and packaged by the sponsor.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Probiotic and prebiotic composition of formulas in the different study groups

 
Measurements
The primary outcome was weight gain in infants fed the study formulas from ≤14 to 112 d of age. The secondary outcomes were recumbent length, head circumference, symptoms of digestive tolerance (GI symptoms, which included frequency of flatulence, colic, spitting up, vomiting, and diarrhea and stool characteristics), and frequency of adverse events (AEs) during the treatment period. Additionally, incidence of diarrhea, antibiotic intake, and hospitalization during the observational period were assessed. Data for the treatment period were based on AE forms filled out by investigators and, for the observational period, on each infant's personal health record book (carnet de santé) completed by the practitioner.

Investigators measured the weight of naked infants on calibrated electronic scales. Measurements were taken twice during each visit (at the beginning and end of the visits) to the nearest 10 g, and the mean was reported. Investigators also measured recumbent length to the nearest 2 mm on standardized length boards and head circumference to the nearest 2 mm with standard measuring tapes. All investigators had the same brand of scales, length boards, and measuring tapes.

Mean formula intake (mL/d) and measurements of digestive tolerance were calculated based on the parents’ records. Digestive tolerance consisted of records of stool frequency and consistency (hard, formed, soft, or liquid); frequency of flatulence (occasional if ≤5/d or often if >5/d); frequency of vomiting and colic (none, once, or more than twice per day); and the occurrence of spitting up. Stool frequency was reported as mean occurrence/d, and all other measurements of tolerance were reported as the percentage of occurrence of a symptom/characteristic. The investigators assessed AEs and the records of digestive tolerance at each visit.

An incident of diarrhea was recorded for infants with at least 1 diarrhea episode. Diarrhea was defined as the occurrence of 3 or more loose or watery stools in 24 h, and an episode of diarrhea was considered to have ended when there were 2 consecutive nonwatery stools or no stools in 24 h.

AEs
AEs were defined as illnesses or signs or symptoms of illnesses (including abnormal laboratory measurements) that occurred or worsened during the course of the study, and they were assessed based on inquiries to the parents and on their daily records. All AEs were recorded and evaluated by the investigators for causality and severity. AEs were assessed as serious if they were life threatening, caused permanent harm, resulted in hospitalization or extension of in-patient hospital treatment, or were considered to be medically relevant by the investigator. All other AEs were categorized as nonserious. AEs were coded using the Medical Dictionary for Regulatory Activities, and analysis was performed for all enrolled subjects.

Statistics
Sample-size calculation was based on the primary outcome, weight gain during the first 4 mo of life (14–112 d). The objective was to show equivalence in weight gain using the {Delta} = 3.9 g/d margin of equivalence (a convention used in previous trials) (18). This is the largest acceptable difference, and any difference greater than this was considered to be clinically relevant. To show weight gain as significant on an {alpha}-level of 5%, the two-sided 90% CI of the treatment difference had to fall entirely in the –3.9 to +3.9 g/d range. The expected SD of weight gain was taken from a previous trial, with {sigma} = 6.1 g/d (18). The sample size was calculated with ni = 55 per group to maintain a power of 90%. Taking a dropout rate of 25% into account, 296 infants had to be enrolled. The control group was made up of infants recruited for this study and was an entirely different group from infants in the study by Puccio et al (18). The sample-size calculation was performed with PASS 2000 (Number Cruncher Statistical Systems, Kaysville, UT).

For the treatment period (0–4 mo), data from all randomly assigned infants were used in the intention-to-treat (ITT) analysis. The per-protocol (PP) analysis also included only data from the treatment period but excluded data from subjects if, during the study, they had a life-threatening event, were hospitalized >3 days, were off the study formula >3 consecutive days, were fed >1 bottle/wk of a different formula, or took complementary food. Primary outcome was analyzed in both the ITT and PP populations. Secondary outcomes were analyzed in the PP population, and safety in the ITT population. For the observational period (4–12 mo), only data from infants who followed up to 12 mo were analyzed.

Changes in weight (weight gain), recumbent length, and head circumference were analyzed by a mixed model correcting for sex and center. The mixed model describes the development of weight, length, and head circumference over time by a quadratic curve, taking into account each subject's intercept and slope (random effects). The mixed model was inquired for the slope in the middle (day 63) of the treatment period (day 14 to day 112). The slope at day 63 equaled the weight gain, calculated by difference in weight divided by the difference in age (from day 14 to day 112). The mixed model was used rather than the analysis of variance (ANOVA) because it does not have the problem of meeting the exact target ages (which varied because of differences in the time of visit); takes into account all weight measurements (at 14, 28, 56, and 112 days); and is robust against dropouts. CI of treatment differences were adjusted for multiple comparisons according to Bretz et al (27). Weight-for-age, length-for-age, and BMI-for-age z-scores were calculated based on the WHO Child Growth Standard (28), which is based on data from exclusively breastfed infants.

Formula intake was analyzed by a mixed model, and a general treatment effect was assessed by a likelihood ratio test. Stool consistency, flatulence, colic, spitting up, vomiting, incidence of diarrhea, antibiotic intake, and hospitalization were analyzed by logistic regression, and treatment effects were presented as odds ratios. Multiple comparisons were adjusted according to Hommel (29). For stool frequency, differences between treatment groups were analyzed by ANOVA and adjusted for multiple tests by Tukey-Kramer. Incidence of infants experiencing at least 1 AE was analyzed by logistic regression, correcting for center.

Age of terminating assigned formulas was analyzed by ANOVA. Treatment differences between control and experimental groups were determined and P values were adjusted according to the Dunnett test. ANOVA and logistic regression were performed with SAS (SAS Institute Inc, Cary, NC) and the mixed models with R 2.1.1 (The R Foundation for Statistical Computing, Vienna, Austria).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subjects
Two-hundred eighty-four healthy infants were enrolled and constituted the ITT population. A total of 57 infants (20%) dropped out by the end of the treatment period (at 4 mo of age), resulting in a PP population of 227. Dropouts were due to change of formula (n = 21), adverse events (n = 5), and loss in follow-up (n = 31) (Figure 1Go). From 4 to 12 mo of age (observational period), an additional 85 infants were lost to follow-up. There were no significant differences in the dropout rates between groups (Kruskal-Wallis test, P > 0.1). Baseline characteristics, other than the mode of birth, were comparable between groups (Table 2Go). More natural births were reported in the BL999 + LPR + GOS/SCFOS group compared with the other groups (P = 0.034).


Figure 1
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 1.. Flow chart of progression of infants during the study.

 

View this table:
[in this window]
[in a new window]

  		TABLE 2. Baseline demographic data of infants by treatment group (per protocol data set n = 227)1

 
Formula intake and growth
Infants in the different groups consumed similar quantities of the study formulas during the treatment period (0–4 mo) (Figure 2Go). The age at which feeding with the assigned formulas was terminated was significantly later in the BL999 + LPR group compared with the control group (mean age 6.56 ± 1.2 mo compared with 5.84 ± 0.7 mo, P = 0.045). However, there were no significant differences between the other study formula groups and the control group.


Figure 2
View larger version (11K):
[in this window]
[in a new window]

  		FIGURE 2.. Volume of daily formula intake (x ± SD) during the treatment period (PP population).

 
Weight gain during the treatment period was equivalent among infants in the different formula groups (Table 3Go). A comparison of the mean weight gain per day of infants fed each of the study formulas with that of infants fed the control formula (PP population) showed a treatment effect of 1.41 (90% CI: –0.77 to 3.59) in the BL999 + LPR group; 0.82 (90% CI: –1.40 to 3.05) in the BL999 + LPR + GOS/SCFOS group; and 1.64 (90% CI: –0.53 to 3.81) in the BL999 + ST11 + GOS/SCFOS group. In all cases the two-sided 90% CI interval lay between –3.9 and + 3.9 g/d, indicating equivalent weight gain in infants fed the control and study formulas. Furthermore, the upper bound of the two-sided 90% CI was above –3 g/d in all comparisons, fulfilling the safety criteria of the American Academy of Pediatrics (30). Analysis of the ITT data set showed similar results (data not shown).


View this table:
[in this window]
[in a new window]

  		TABLE 3. Mean (±SD) weight gain and changes in length, head circumference, and body mass index (BMI) during the treatment period (0–4 mo) in the per protocol population1

 
Mean changes in length, head circumference, and BMI measurements during the treatment period were not different between all of the study formula groups and the control group (P > 0.05 for all comparisons, Table 3Go). A comparison of weight and length z-scores with the WHO multicenter growth reference (28) showed that, among infants who completed the study to 12 mo, z-scores were close to 0 at all times during the study (Figure 3Go). Z-scores were not different from the WHO growth reference for all groups in the PP population followed through the treatment period (0–4 mo), and no significant differences were observed between the control and any of the study formula groups (data not shown). Comparison with Euro-growth reference for head circumference (31, 32) (WHO standards for head circumference were unavailable) also showed that z-scores were not different from the standard (data not shown).


Figure 3
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 3.. Weight- and length-for-age z-scores (x ± SD), relative to WHO standards, of the subset of infants who completed the study to 12 mo. Treatment period of 2–16 wk and observation period of 52 wk.

 
Safety and tolerance
The incidence of diarrhea during the treatment period was not different between the control and study formula groups (Table 4Go). However, during the observation period, the incidence of diarrhea was significantly lower in the BL999 + LPR group compared with the control group (5/37 compared with 13/30). The frequency of antibiotic treatment or hospitalization during this period did not vary among groups (Table 4Go).


View this table:
[in this window]
[in a new window]

  		TABLE 4. Incidence (i) of diarrhea during the treatment period (0–4 mo) in the ITT population and during the observational period (4–12 mo) in infants who were followed up to 1 y

 
During the treatment period, stool frequency was significantly higher among infants in the BL999 + LPR + GOS/SCFOS group compared with those in the control group (x frequency 2.1/d compared with 1.6/d, P = 0.03). Although there was not a significant difference in stool frequency between the other study formula groups and the control group, infants in the BL999 + ST11 + GOS/SCFOS group tended to have higher but not significantly different stool frequencies than those in the control group (mean 2.0 versus 1.6/d, P = 0.06). Furthermore, infants in the BL999 + LPR + GOS/SCFOS group had higher stool frequency compared with those in the BL999 + LPR group, but this difference was not significant (x 2.1/d compared with 1.7/d, P = 0.07).

During the treatment period, liquid stools occurred significantly more frequently in the BL999 + ST11 + GOS/SCFOS group than in both the control group (odds ratio = 3.17, 95% CI: 1.59 to 6.30, P = 0.005) and the BL999 + LPR group (odds ratio = 2.79, 95% CI: 1.48 to 5.29, P = 0.008). The difference in the frequency of liquid stools between any of the other groups was not significant. Furthermore, the frequency with which the other stool consistencies (soft, formed, hard) occurred in all groups were not significantly different. The frequency with which flatulence, colic, spitting up, and vomiting occurred were not significantly different in the control and study formula groups (P > 0.1 for all).

The occurrence of at least 1 serious AE (SAE) was reported in 24 infants (Table 5Go), and at least 1 AE was reported in 184 infants during treatment period. There were no significant differences in the frequency of either SAEs or AEs between the different groups. Two SAEs involving cow's milk protein allergy was assessed as certainly and possibly related to the study formula (both in the BL999 + LPR group). Two others, a case of diarrhea and a case of gastroesophageal reflux disease, were considered to be probably related to the formula (both in the control group). The rest of the SAEs (except 4 whose causality were not assessed by the investigators) were considered to be unrelated or unlikely to be related to the study products. Most (78%) of the AEs were respiratory and GI problems (including allergies) and infections.


View this table:
[in this window]
[in a new window]

  		TABLE 5. Number (%) of serious adverse events (SAE) presented by preferred term based on coding by MedDRA

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we evaluated the safety of 3 infant formulas that were supplemented with probiotics or synbiotics. The primary outcome, weight gain following 14–16 wk of formula feeding (treatment period), was equivalent in the control and study formula groups. Similarly, changes in length, head circumference, and BMI between 0 and 4 mo of age were similar in all study groups. Employing additional measures of safety and tolerance, we also evaluated the occurrence of various GI symptoms, including diarrhea and the need for antibiotic treatment or hospitalization during the treatment (0–4 mo) and observational (4–12 mo) periods. During both periods infants in all groups grew normally; weight-for-age and length-for-age z-scores indicated growth rates comparable to WHO growth standards. Because the WHO growth standards are based on healthy, exclusively breastfed infants, these results are a good indication of the nutritional sufficiency of these formulas, though no direct comparison of the value of these formulas with breastfeeding can be made based on our data. Although not statistically significant, the differences in z-scores for length at 12 mo suggest that there might be a difference in the effect of the 2 formulas containing LPR compared with the control. However, our study was not designed to detect this difference, and future studies with significantly more subjects (424 per group to see a difference of 0.87 mm/mo, the difference between the BL999 + LPR and control groups) will have to be performed to determine whether there is a real effect of LPR on length. Furthermore, all of the comparisons in weight gain between study formula and control groups showed a trend toward greater weight gain in the former. To see a difference in weight-for-age z-scores of 0.1 (which was the treatment difference between BL999 + LPR and control group after 1 year) as significant with a power of 80%, a new trial with 2654 infants per group would have to be conducted. A recent study (33) specifically evaluating the growth of infants fed formula containing L. rhamnosus has shown that these infants gained weight at a significantly faster rate compared with those who were fed a control formula. Whether L. rhamnosus leads to increased weight gain needs to be confirmed, but these results indicate its safety in healthy infants.

Interestingly, whereas during the treatment period there were no differences in the frequency of symptoms of GI intolerance, diarrhea, and other AEs between infants in any of the study formula groups and those in the control formula group, at the 1-year follow-up, infants in the group that received the formula containing B. longum BL999 and L. rhamnosus LPR (BL999 + LPR) had significantly fewer incidents of diarrhea. It is intriguing and, to our knowledge, novel that the decrease in the incidence of diarrhea was observed several months after infants had stopped taking the probiotic-supplemented formula. Even though infants in the BL999 + LPR group continued to take their assigned formula longer than infants in the control group, they had stopped taking the probiotic-supplemented formula by the time they were, on average, 6.5 ± 1.2 mo old. Therefore, this finding suggests that the effect of the probiotics in this group may have lasted an average of 5.5 ± 0.7 mo after the termination of administration. Because other possible confounding factors during the observational period (such as diet) were not studied here, it is not possible to attribute our observation solely to the effect of probiotics.

There are numerous studies demonstrating the efficacy of probiotics in treating or preventing diarrhea (6, 7, 9, 20, 21), including during antibiotic treatment (34). However, the effect of probiotics following an extended period after intake has been terminated has not yet been reported. Although our results will have to be confirmed in future studies aimed specifically at addressing the long-term effects of probiotics (including microbiological analyses of stool samples during treatment and control for confounding factors), they do suggest that when intake of probiotics occurs early in an infant's life (2 wk or younger), there may be a long-term beneficial effect on the incidence of diarrhea. It is interesting to speculate that administering probiotics very early in the infants’ lives, when their GI microbiota and immune system were still not fully developed, may have influenced their microbiota and primed their immune system. By being recognized as "nonself" by the immune system, probiotics could have acted as adjuvants, increasing the mucosal immune system activity and natural defenses, thereby affecting health at a later period. Alternatively, administering probiotics at this early stage may have influenced the development of the gut immune system and the maturation of the gut itself, which may then have contributed to the improved ability of infants to resist diarrhea later on. Unfortunately, microbiological analysis was not performed in this study so we cannot begin to address these possibilities here, but it will be an important aspect of future investigations. It is surprising that a similar effect among infants in the BL999 + LPR + GOS/SCFOS group was not observed. This finding may suggest a specific effect of either or both probiotic strains (B. longum BL999 or L. rhamnosus LPR). We hypothesize that in the BL999 + LPR + GOS/SCFOS group, the prebiotics mixture may have dampened the effect of the probiotics seen in the BL999 + LPR group by stimulating the growth of other components of the microbiota, thereby effectively reducing the relative proportion of the probiotics among the GI microbiota or hiding the role of a probiotic in the imprinting of the neonatal immune system. A recent publication by Ziegler et al (35) showed that a formula supplemented only with prebiotics led to significantly higher incidence of diarrhea in the short term (during 30–120 d of feeding) compared with an unsupplemented formula. This finding suggests that supplementation with specific probiotics may be more beneficial than stimulating the growth of a broader population of bacteria.

The tendency for infants fed prebiotics to have an increase in overall stool frequency and in the frequency of liquid stools, though statistically significant, bears little clinical relevance because these frequencies were all within the normal range for infants. Previous studies have shown that GOS/FOS causes a slight increase in stool frequency (36, 37) and frequency of loose stools (18, 35, 37, 38). However, it was also shown that there was no correlation between the increase in stool frequency and lower weight gain in at least 1 study (18).

Thus, this study confirms the safety of different mixtures of probiotics and synbiotics. It also raises the possibility that the specific mixture containing B. longum BL999 and L. rhamnosus LPR may have a prolonged effect in reducing the incidence of diarrhea.


   ACKNOWLEDGMENTS
 
We thank Makda Fisseha for providing medical writing services on behalf of HPM Geneva SA. We also thank G. Putet (Lyon), C. Hubert (Nancy), F Gold, C Grillon (Paris), U. Simeoni (Marseille), and all residents and medical staff of each team for their contribution to the study.

The authors’ responsibilities were as follows—JPC: main investigator and coordinator of the study; JML, JMH, IdM, ML: co-investigators; PS: study sponsor and coordinator; MG: clinical project manager; DG: performed the statistical analysis and was involved in the interpretation of the results. DG, MG, and PS are employed by Nestlé, the sponsoring company. None of the other authors has a conflict of interest to disclose.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Newburg DS. Innate immunity and human milk. J Nutr 2005;135:1308–12.[Abstract/Free Full Text]
  2. Balmer SE, Wharton BA. Diet and faecal flora in the newborn: breast milk and infant formula. Arch Dis Child 1989;64:1672–7.[Abstract/Free Full Text]
  3. Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, et al. Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr 2000;30:61–7.[Medline]
  4. Mountzouris KC, McCartney AL, Gibson GR. Intestinal microflora of human infants and current trends for its nutritional modulation. Br J Nutr 2002;87:405–20.[Medline]
  5. Newburg DS. Oligosaccharides in human milk and bacterial colonization. J Pediatr Gastroenterol Nutr 2000;30(suppl 2):S8–17.[Medline]
  6. Chouraqui JP, Van Egroo LD, Fichot MC. Acidified milk formula supplemented with bifidobacterium lactis: impact on infant diarrhea in residential care settings. J Pediatr Gastroenterol Nutr 2004;38:288–92.[Medline]
  7. Weizman Z, Asli G, Alsheikh A. Effect of a probiotic infant formula on infections in child care centers: comparison of two probiotic agents. Pediatrics 2005;115:5–9.[Abstract/Free Full Text]
  8. Saavedra JM, Bauman NA, Oung I, Perman JA, Yolken RH. Feeding of Bifidobacterium bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus. Lancet 1994;344:1046–9.[Medline]
  9. Szajewska H, Mrukowicz JZ. Probiotics in the treatment and prevention of acute infectious diarrhea in infants and children: a systematic review of published randomized, double-blind, placebo-controlled trials. J Pediatr Gastroenterol Nutr 2001;33(suppl 2):S17–25.[Medline]
  10. Marteau PR, de Vrese M, Cellier CJ, Schrezenmeir J. Protection from gastrointestinal diseases with the use of probiotics. Am J Clin Nutr 2001;73(suppl):S430–6.[Abstract/Free Full Text]
  11. Kalliomaki M, Salminen S, Arvilommi H, Kero P, Koskinen P, Isolauri E. Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial. Lancet 2001;357:1076–9.[Medline]
  12. Isolauri E, Arvola T, Sutas Y, Moilanen E, Salminen S. Probiotics in the management of atopic eczema. Clin Exp Allergy 2000;30:1604–10.[Medline]
  13. Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr 1995;125:1401–12.[Abstract/Free Full Text]
  14. Cummings JH, Macfarlane GT. The control and consequences of bacterial fermentation in the human colon. J Appl Bacteriol 1991;70:443–59.[Medline]
  15. Sako T, Matsumoto K, Tanaka R. Recent progress on research and applications of non-digestible galacto-oligosaccharides. Int Dairy J 1999;9:69–80.
  16. Chonan O, Takahashi R, Watanuki M. Role of activity of gastrointestinal microflora in absorption of calcium and magnesium in rats fed β1–4 linked galactooligosaccharides. Biosci Biotechnol Biochem 2001;65:1872–5.[Medline]
  17. Collins MD, Gibson GR. Probiotics, prebiotics, and synbiotics: approaches for modulating the microbial ecology of the gut. Am J Clin Nutr 1999;69(suppl):S1052–7.[Medline]
  18. Puccio G, Cajozzo C, Meli F, Rochat F, Grathwohl D, Steenhout P. Clinical evaluation of a new starter formula for infants containing live Bifidobacterium longum BL999 and prebiotics. Nutrition 2007;23:1–8.[Medline]
  19. Isolauri E, Juntunen M, Rautanen T, Sillanaukee P, Koivula T. A human Lactobacillus strain (Lactobacillus casei sp strain GG) promotes recovery from acute diarrhea in children. Pediatrics 1991;88:90–7.[Abstract/Free Full Text]
  20. Katz JA. Probiotics for the prevention of antibiotic-associated diarrhea and Clostridium difficile diarrhea. J Clin Gastroenterol 2006;40:249–55.[Medline]
  21. Kullen MJ, Bettler J. The delivery of probiotics and prebiotics to infants. Curr Pharm Des 2005;11:55–74.[Medline]
  22. Akiyama K, Shimada M, Ishizeki S, et al. Effects of administration of Bifidobacterium in extremely premature infants; development of intestinal microflora by orally administered Bifidobacterium longum in comparison with Bifidobacterium breve. Acta Neonatol Jpn 1993;30:257–63.
  23. Bennet R, Nord CE, Zetterstrom R. Transient colonization of the gut of newborn infants by orally administered bifidobacteria and lactobacilli. Acta Paediatr 1992;81:784–7.[Medline]
  24. Xiao JZ, Kondo S, Yanagisawa N, et al. Effect of probiotic Bifidobacterium longum BB536 [corrected] in relieving clinical symptoms and modulating plasma cytokine levels of Japanese cedar pollinosis during the pollen season. A randomized, double-blind, placebo-controlled trial. J Investig Allergol Clin Immunol 2006;16:86–93.[Medline]
  25. Cruchet S, Obregon MC, Salazar G, Diaz E, Gotteland M. Effect of the ingestion of a dietary product containing Lactobacillus johnsonii La1 on Helicobacter pylori colonization in children. Nutrition 2003;19:716–21.[Medline]
  26. Sarker SA, Sultana S, Fuchs GJ, et al. Lactobacillus paracasei strain ST11 has no effect on rotavirus but ameliorates the outcome of nonrotavirus diarrhea in children from Bangladesh. Pediatrics 2005;116:e221–8.[Abstract/Free Full Text]
  27. Bretz F, Genz A, Hothorn L. On the numerical availability of multiple comparison procedures. Biometrical J 2005;43:645–56.
  28. WHO Multicenter Growth Reference Study Group. WHO Child Growth Standards: length/height-for-age, weight-for-age, weight-for-length, weight-for-height and body mass index-for-age: methods and development. World Health Organization, Geneva, 2006. http://www.who.int/nutrition/media_page/en/ (accessed November 2006).
  29. Hommel GA. A stagewise rejective multiple test procedure based on a modified Bonnferroni test. Biometrika 1988;75:383–6.[Abstract/Free Full Text]
  30. U.S. Food and Drug Administration/American Academy of Pediatrics Committee on Nutrition. Clinical testing of infant formulas with respect to nutritional suitability for term infants. http://www.cfsan.fda.gov/~dms/inf-clin.html; 1988 (accessed 2003).
  31. Haschke F, van't Hof MA. Euro-Growth references for length, weight, and body circumferences. Euro-Growth Study Group. J Pediatr Gastroenterol Nutr 2000;31(suppl1):S14–38.[Medline]
  32. van't Hof MA, Haschke F. Euro-Growth references for body mass index and weight for length. Euro-Growth Study Group. J Pediatr Gastroenterol Nutr 2000;31(suppl1):S48–59.[Medline]
  33. Vendt N, Grunberg H, Tuure T, et al. Growth during the first 6 months of life in infants using formula enriched with Lactobacillus rhamnosus GG: double-blind, randomized trial. J Hum Nutr Diet 2006;19:51–8.[Medline]
  34. Correa NB, Peret Filho LA, Penna FJ, Lima FM, Nicoli JR. A randomized formula controlled trial of Bifidobacterium lactis and Streptococcus thermophilus for prevention of antibiotic-associated diarrhea in infants. J Clin Gastroenterol 2005;39:385–9.[Medline]
  35. Ziegler E, Vanderhoof JA, Petschow B, et al. Term infants fed formula supplemented with selected blends of prebiotics grow normally and have soft stools similar to those reported for breast-fed infants. J Pediatr Gastroenterol Nutr 2007;44:359–64.[Medline]
  36. Boehm G, Lidestri M, Casetta P, et al. Supplementation of a bovine milk formula with an oligosaccharide mixture increases counts of faecal bifidobacteria in preterm infants. Arch Dis Child Fetal Neonatal Ed 2002;86:F178–81.[Abstract/Free Full Text]
  37. Moro G, Minoli I, Mosca M, et al. Dosage-related bifidogenic effects of galacto- and fructooligosaccharides in formula-fed term infants. J Pediatr Gastroenterol Nutr 2002;34:291–5.[Medline]
  38. Euler AR, Mitchell DK, Kline R, Pickering LK. Prebiotic effect of fructo-oligosaccharide supplemented term infant formula at two concentrations compared with unsupplemented formula and human milk. J Pediatr Gastroenterol Nutr 2005;40:157–64.[Medline]
Received for publication July 31, 2007. Accepted for publication December 16, 2007.




This article has been cited by other articles:


Home page
 Appl. Environ. Microbiol.Home page
C. Devirgiliis, D. Coppola, S. Barile, B. Colonna, and G. Perozzi
Characterization of the Tn916 Conjugative Transposon in a Food-Borne Strain of Lactobacillus paracasei
Appl. Envir. Microbiol., June 15, 2009; 75(12): 3866 - 3871.
[Abstract] [Full Text] [PDF]

Home page
 Nutr Clin PractHome page
R. J. Joeckel and S. K. Phillips
Overview of Infant and Pediatric Formulas
Nutr Clin Pract, June 1, 2009; 24(3): 356 - 362.
[Abstract] [Full Text] [PDF]

Home page
 Nutr Clin PractHome page
A. Minocha
Probiotics for Preventive Health
Nutr Clin Pract, April 1, 2009; 24(2): 227 - 241.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chouraqui, J. P.
Right arrow Articles by Steenhout, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chouraqui, J. P.
Right arrow Articles by Steenhout, P.
Agricola
Right arrow Articles by Chouraqui, J. P.
Right arrow Articles by Steenhout, P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS