Oral Probiotics Prevent Necrotizing Enterocolitis in Very Low Birth Weight Preterm Infants: A Multicenter, Randomized, Controlled Trial
OBJECTIVE. The goal was to investigate the efficacy of orally administered probiotics in preventing necrotizing enterocolitis for very low birth weight preterm infants.
METHODS. A prospective, blinded, randomized, multicenter controlled trial was conducted at 7 NICUs in Taiwan, to evaluate the beneficial effects of probiotics in necrotizing enterocolitis among very low birth weight infants (birth weight: <1500 g). Very low birth weight infants who survived to start enteral feeding were eligible and were assigned randomly to 2 groups after parental informed consent was obtained. Infants in the study group were given Bifidobacterium bifidum and Lactobacillus acidophilus, added to breast milk or mixed feeding (breast milk and formula), twice daily for 6 weeks. Infants in the control group were fed with breast milk or mixed feeding. The clinicians caring for the infants were blinded to the group assignment. The primary outcome measurement was death or necrotizing enterocolitis (Bell's stage ≥2).
RESULTS. Four hundred thirty-four infants were enrolled, 217 in the study group and 217 in the control group. The incidence of death or necrotizing enterocolitis (stage ≥2) was significantly lower in the study group (4 of 217 infants vs 20 of 217 infants). The incidence of necrotizing enterocolitis (stage ≥2) was lower in the study group, compared with the control group (4 of 217 infants vs 14 of 217 infants). No adverse effect, such as sepsis, flatulence, or diarrhea, was noted.
CONCLUSION. Probiotics, in the form of Bifidobacterium and Lactobacillus, fed enterally to very low birth weight preterm infants for 6 weeks reduced the incidence of death or necrotizing enterocolitis.
Necrotizing enterocolitis (NEC) is one of the most catastrophic gastrointestinal emergencies in very low birth weight (VLBW) preterm infants, affecting 7% to 14% of these infants.1 NEC is a leading cause of death and morbidity in NICUs,2,3 and the incidence of NEC has not changed in the past 20 years.2 Recent reports suggested the increasing occurrence of NEC and estimated up to 9000 cases of NEC in the United States every year, with a case fatality rate of 15% to 30%.4
The pathogenesis of NEC remains an enigma, but it is widely considered a multifactorial disease; prematurity, enteral feeding, intestinal hypoxia-ischemia, and bacterial colonization are considered major risk factors. Most likely, NEC is the clinical culmination of multiple risk factors that result in bowel injury through a final, common, inflammatory pathway.5–8
It has been suggested that an inappropriate, accentuated, inflammatory response to colonizing pathogenic florae in the premature gastrointestinal tract plays a major role in the initiation of NEC.9 The inflammatory cascade promotes the spread of bacteria or toxin, resulting in ischemia, necrosis, and, in some cases, perforation.10 In vitro evidence showed that pathogenic florae attach to the epithelial cells of preterm infants much more easily than to those of term infants,11,12 and studies indicated that commensal bacteria could inhibit or reduce inflammatory signaling in intestinal epithelia through inhibition of the NF-κB pathway.13,14 These data suggest that probiotics, by modifying the occurrence of these cascades of events, may play a major role in reducing the incidence of NEC.
Bin-Nun et al15 and we16 showed that orally administered probiotics reduce the incidence of NEC in VLBW preterm infants. Two meta-analyses arrived at the same conclusion; however, the limited number of clinical trials results in lack of definition of optimal strains, timing, dosage, and duration of probiotics administered to VLBW preterm infants, and these issues need to be evaluated in large trials.17,18 Furthermore, it has been speculated that the microbes of the developing intestinal tract of premature infants affect the maturation and optimal functioning of the intestinal innate and adaptive immune systems. Therefore, intestinal microbes may be involved in the pathogenesis of systemic inflammation-related diseases such as chronic lung disease (CLD), intraventricular hemorrhage (IVH), and periventricular leukomalacia (PVL).19,20 However, no clinical trial has examined this hypothesis.
Bifidobacteria and lactobacilli are commonly found in breastfed infants,21 and most cases of NEC in VLBW infants occur before 6 weeks of age.22 We hypothesized that orally administered probiotics, in the form of bifidobacteria and lactobacilli, fed to VLBW preterm infants for 6 weeks would reduce the incidence and severity of NEC.
We performed a pilot study from January 1, 2005, to March 31, 2005, to verify the viability of probiotic bacteria in the stool when Infloran (National Collection of Dairy Organisms, Reading, United Kingdom and Laboratorio, Farmaceutico, Mede, Italy)(containing Lactobacillus acidophilus and Bifidobacterium bifidum) was fed to VLBW preterm infants. Stool was collected for the first week of life from VLBW preterm infants (gestational age: <34 weeks; birth weight: <1500 g) who were fed Infloran in the NICU of China Medical University Hospital, after informed parental consent was obtained. Fecal samples were analyzed by using the methods reported by Lee et al23; we confirmed that B bifidum and L acidophilus colonized the intestines of preterm infants fed Infloran.
From April 1, 2005, to May 30, 2007, a prospective, masked, randomized, controlled trial was conducted in 7 level III NICUs in Taiwan. The study protocol was approved by the institutional review board of each hospital. VLBW preterm infants (gestational age: <34 weeks; birth weight: <1500 g) who survived to feed enterally were eligible for the trial. They were assigned randomly to the study or control group by the principal investigator at each center after informed parental consent was obtained. Randomization was performed by using sequential numbers generated at the computer center of China Medical University Hospital and sent to the principal investigator at each center when an infant was eligible for enrollment. VLBW preterm infants who had severe asphyxia (stage III), fetal chromosomal anomalies, cyanotic congenital heart disease, congenital intestinal atresia, gastroschisis, or omphalocele, those who were fed exclusively with formula, and those who were fasted for >3 weeks were excluded.
The study group was given Infloran (L acidophilus [109 colony-forming units, NCDO 1748; National Collection of Dairy Organisms] and B bifidum [109 colony-forming units, NCDO 1453; National Collection of Dairy Organisms, Reading, United Kingdom]; Laboratorio Farmaceutico, Italy) at 125 mg/kg per dose twice daily, through addition to breast milk or mixed feeding (breast milk and formula), for 6 weeks; the control group was fed breast milk or mixed feeding. Infloran was sent to each center and stored in a refrigerator at 2°C to 8°C. Infloran was added to breast milk (the infant's own mother's milk) or formula by the breast milk team before feeding. Both breast milk and formula for the study and control groups were prepared by the breast milk team, who did not know the colony counts of probiotics and were not involved in the care of the infants. The team members followed the orders from a sealed envelope. Therefore, the only personnel who knew of the infants' group assignments were the investigators at each center and those on the breast milk team, who were not involved in the care of the study infants.
The indications for feeding and a strict feeding protocol were followed for all study infants, as described in a previous study.16 Depending on the birth weight and gestational age, a certain amount of breast milk was initiated after the infant tolerated 1 trial of distilled water. On the first day, 1 mL/kg distilled water was given twice, followed by breast milk. The amount of feeding was increased slowly if tolerated, with increments of no more than 20 mL/kg per day per feeding. An oral intake of 100 mL/kg per day was defined as complete enteral feeding. Feeding was stopped if there was any sign of feeding intolerance, defined as the presence of gastric aspirate in an amount that was more than one half of the previous feeding, twice, with abdominal distension. Infants who weighed <1000 g received total parenteral nutrition until one half of their energy was supplied through the oral route. The same attending physician was in charge of the care of the infants during their hospital stay. The residents who rotated through the NICU provided care by following established protocols in the unit. Definitions of prenatal steroid use, small for gestational age, prolonged rupture of amniotic membranes, chorioamnionitis, asphyxia, respiratory distress syndrome, patent ductus arteriosus, IVH, PVL, and sepsis and indications for surfactant and indomethacin were as described in our previous study.15 The management protocols, clinical practices, equipment, infrastructure, and key personnel in each unit were unchanged during the study period. A consensus was obtained among the 7 participating centers regarding the definition and prospective collection of demographic and clinical outcome variables. The data collected by each center were transmitted to the office of the principal investigator (Dr Lin) at China Medical University Hospital.
NEC was diagnosed and classified according to a modification of the classification described by Bell et al.24 The final determination of the diagnosis of NEC of stage ≥2 was made by 2 independent attending physicians who did not know the group assignment of the infant. If there was disagreement, then the senior neonatologist in each center was consulted to make the final diagnosis. Spontaneous intestinal perforation was diagnosed on the basis of clinical, radiologic, and histologic findings25 by 2 independent attending physicians who did not know the group assignment of the infant.
Demographic and clinical variables that were potential risk factors for NEC were recorded in the protocol once the infants completed the 6-week study period. The primary outcome was death or stage ≥2 NEC that occurred during the study period. Secondary outcomes were culture-proven sepsis without NEC, CLD, IVH, PVL, feeding amount per week, days to full enteral feeding, and weight gain per week. Adverse effects, including culture-proven sepsis attributable to L acidophilus or B bifidum, flatulence, and diarrhea, also were recorded.
The event rate for sample size calculation was based on unpublished data of the Premature Infant Foundation of Taiwan and the Bravura National Health Insurance database (2003–2004). Their recent event rate for death or NEC (stage ≥2) for VLBW preterm infants was ∼25% for level III neonatal centers in Taiwan. With the α error set at .05 and the β error at .1 and an absolute reduction in the incidence of NEC or death of 50%, the number needed to verify our hypothesis was 203 infants for each arm. Student's 2-tailed t test and the χ2 test or Fisher's exact test were used to explore the differences in continuous and categorical variables, respectively. Univariate and multivariate logistic regression models were used to estimate odds ratios and 95% confidence intervals. Multivariate logistic regression analysis was used to assess the relationship between different outcome measures and the effect of probiotics, after adjustment for the potentially confounding effects of demographic factors, clinical characteristics, and possible bias attributable to the multicenter design. All statistical tests were 2-sided, and a P value of <.05 was considered to indicate statistical significance. Analyses were performed with SAS 9.1 (SAS Institute, Cary, NC).
Figure 1 shows the flow of study subjects through the phases of the study. There were 580 VLBW infants admitted to the 7 NICUs during the study period. One hundred thirty-seven infants were excluded; either they died (n = 98), they met the exclusion criteria (n = 14), or family members declined consent for the study (n = 25). A total of 443 infants were enrolled in the trial; 3 infants in the study group and 1 in the control group dropped out of the program because of parental wishes to withdraw the infants from the trial. Two infants in the study group and 3 in the control group underwent spontaneous intestinal perforation and were excluded. There were 217 infants in the study arm and 217 in the control arm. The mothers' clinical and infants' demographic and clinical characteristics did not differ between the 2 groups, except for lower birth weight and first arterial blood pH values in the study group (Table 1). The infants' clinical variables also did not differ between the 2 groups, except for more frequent use of surfactant replacement therapy in the study group (Table 2).
Table 3 shows the primary outcomes of the study. The incidence of death or stage ≥2 NEC (4 of 217 infants vs 20 of 217 infants; P = .002) was significantly lower in the study group, compared with the control group. Two infants in the study group and 9 infants in the control group developed stage 2 NEC; 2 infants in the study group and 5 infants in the control group developed stage 3 NEC. Four and 14 infants developed stage ≥2 NEC in the study and control groups, respectively (P < .02). There was no difference between the study group and control group in the incidence of death attributable to NEC. The incidence of death not attributable to NEC (0 of 217 infants vs 6 of 217 infants; P = .04) was significantly lower in the study group, compared with the control group.
The outcomes of infants enrolled in each center are shown in Table 4. Table 5 shows the unadjusted and adjusted odds ratios for primary (death or NEC) and secondary outcome variables. The incidence of death or stage ≥2 NEC was significantly lower in the probiotic-treated group in both unadjusted and adjusted analyses; the latter was performed by entering potential confounding variables (birth weight, surfactant use, pH, gestational age, and center) into the multivariate logistic regression model. Similar results were found in the analysis of NEC alone. For the secondary outcomes, there were no differences between the 2 groups with respect to PVL and severe IVH; although sepsis was more frequent among infants in the study group, there was no difference in the univariate analysis or with adjustment for various confounding variables, such as birth weight, umbilical venous catheter use, intermittent mandatory ventilation, NICU stay, and center. The pathogens were most often related to catheter-related infections in both groups. None of the positive blood cultures yielded Lactobacillus or Bifidobacterium spp. The incidence of CLD was higher in the study group according to the univariate analysis, but no difference was observed in the logistic regression analysis. No other adverse effect, such as flatulence or diarrhea, was noted.
Table 6 presents outcome variables for different weight groups in the study and control groups. The number of infants was limited for infants weighing <750 g, and results are difficult to interpret because of type II error. Incidence of death or NEC ≥ stage 2 was significantly lower in infants weighted 500–750 g (p = 0.02) and 1001–1500g (p = 0.02), but approach to nearly significant in infants weighted 751–1000 g (p = 0.07). Incidence of NEC ≥ stage 2 was significantly lower in infants weighted 1001–1500 g (p = 0.02), but not significant in infants weighted 500–750 g and 751–1000 g. There was a trend that incidence NEC ≥ stage 2 was lower in infants weighted 500–750 g and 751–1000 g (1 vs 2 and 3 vs 5). Occurrence of sepsis was most often in the infants weighted <1000 g. The incidence of Gram-positive and Gram-negative sepsis did not differ between study and control groups for infants in the different weight groups.
Table 7 shows that the age of attainment of full feeding and weight gain at various ages were similar between the 2 groups.
This is the first multicenter, randomized, controlled trial that showed efficacy of probiotics containing B bifidum and L acidophilus in reducing the incidence of NEC among VLBW preterm infants. We also found that the study group had a lower incidence of death or NEC. According to our data, the number needed to treat to prevent 1 case of NEC was 20 patients, and the number needed to treat to prevent 1 death or case of NEC was 14 patients.
Many variables have been suggested to be associated with the development of NEC; however, only prematurity and low birth weight have been consistently identified in case-control studies.26,27 The current study was designed as a multicenter, randomized, controlled trial. During the randomization process, we neglected to stratify according to birth weight, which resulted in an imbalance of patient assignment to the study and control groups, with the birth weight being lower in the former group. The lower birth weight also accounts for the greater acuity of the infants' illness, as evidenced by lower first arterial pH and more frequent use of surfactant therapy. The difference in birth weight was because there were 33 infants in the study group but only 18 infants in the control group who weighed <750 g. However, in the study group, there was lower incidence of death or NEC in different weight groups; incidence NEC tended to be lower in each weight group and no infants died with NEC. We addressed this methodologic deficiency by performing posthoc, multivariate, logistic regression analysis, entering potential risk factors that could bias the results (eg, birth weight and first arterial pH). Additional support for the validity of our data was the demonstration that the incidence of the primary study outcome (death or NEC) remained lower in the probiotic-treated group. Because center variation can play an important role in outcome, we included center outcomes in the model.
Because of the methodologic misstep that caused uneven distribution of the weight groups and resulted in fewer infants weighing <750 g in the control group, it is difficult to interpret the results for infants weighing <750 g because of type II error. In the study group, however, there were significantly lower incidences of death or NEC in different weight groups; the incidence of NEC tended to be lower in each weight group, and no infants died as a result of causes other than NEC. Additional research with an adequate number of weight-stratified infants is urgently needed to help define the beneficial effects of probiotics in each group.
The ability of bacteria to cross epithelial cell layers is thought to be a crucial first step in the cascade of events leading to the development of NEC.28,29 Bacterial interactions with the premature gut might play a major role in the proposed pathogenesis of NEC; many studies suggest a strong relationship between delay and low colonization of commensal florae and proliferation of pathogenic florae in the immature gut, predisposing preterm infants to develop NEC.28–32 Using animal models, Caplan et al33 and Butel et al34 showed that bifidobacterial supplementation in rat pup and quail models resulted in intestinal colonization and subsequent reduction in NEC-like lesions. Our findings of a beneficial effect of oral probiotic supplementation may be based on this mechanism. Bifidobacteria and lactobacilli have been shown to inhibit intestinal colonization of pathogenic microorganisms, to produce protective nutrients, and to prevent translocation of other bacteria.35 These characteristics support the use of Bifidobacterium and Lactobacillus as appropriate species of probiotics for the prevention of NEC.
The probiotics we used in the current study were different from those in our previous study, because the supplier of the probiotics altered the formula by changing Bifidobacterium infantis (used in our previous trial) to B bifidum (used in current study). In the study by Bin-Nun et al,15 the probiotics contained B infantis, Streptococcus thermophilus, and B bifidum. Molecular studies have indicated that Bifidobacterium spp in the intestinal tract can range from 60% to 90% of the total fecal microbiota in breastfed infants, and lactic acid-producing bacteria may account for <1% of the total microbiota, indicating the significant dominance of Bifidobacterium.36 Studies also indicated that B bifidum, Bifidobacterium longum, and Bifidobacterium breve are the most common strains in healthy breastfeeding infants.37,38 A recent study showed that B bifidum is a promising candidate for probiotic intervention in inflammatory disorders of the gastrointestinal tract.39 On the basis of these clinical trials and in vitro studies, it is reasonable to speculate that probiotics that contain Bifidobacterium might be most appropriate for the prevention of NEC.
Human milk feeding has been shown to reduce the incidence of NEC but cannot eradicate NEC, as in this study. Three of 4 infants in the study group and 6 of 14 infants in the control group were receiving exclusive breast milk feeding but still developed NEC. It has been shown that human milk feeding may not eliminate NEC because of interleukin 10 deficiency.40 Both Bifidobacterial and Lactobacillus has been shown to induce IL10 production.41,42 Breastfeeding promotes a strong bifidobacterial presence in the infant gut by providing oligosaccharides that act as favorable substrates for bifidobacteria. Oral administration of specific strains of Lactobacillus species stimulate the Bifidobacterium microbiota.43 Probiotics and human milk may have synergistic effects with bifidobacteria and lactobacilli to inhibit the inflammatory response in NEC. In the current study and as in most NICUs, a large majority of infants were fed human milk; therefore, it was difficult to demonstrate potential synergistic effects of probiotics and human milk.
Our previous study16 and other studies44,45 showed that probiotics may reduce the incidence of sepsis; however, meta-analysis did not confirm this association.17,18 The current study did not show that probiotics reduced the incidence of sepsis in VLBW infants as in our previous study, and occurrences of sepsis even seemed more frequent in the study group. Our previous study did not examine sepsis in detail. We analyzed the frequency of sepsis according to Gram-positive, Gram-negative, and fungal infections and found that the pathogens were most often related to catheter-related infections in both groups. Theoretically, changing the intestinal ecosystem could not prevent Gram-positive sepsis. The incidence of Gram-negative sepsis was higher in the study group, but no difference was observed in either univariate analysis or logistic regression analysis with risk factors. It is well known that risk factors for late-onset sepsis include young gestational age, use of a central line, total parenteral nutrition, and prolonged use of mechanical ventilation, among others.45 We speculate that probiotics would not prevent late-onset sepsis because of the complexity of this disorder. Probiotics alone could not overcome the invasive procedures inducing infection. The same speculation can be made regarding the nonsignificant effects on CLD, IVH, and PVL. The primary effect of orally administered probiotics is in the gastrointestinal tract. It is not surprising to see the lack of beneficial effects on other organs, such as the lung and central nervous system.
A few case reports have raised concerns regarding infections with probiotic microorganisms in patients who are immunocompromised or have underlying medical conditions predisposing them to infection.47,48 However, it was noted by Presterl et al48 that some Lactobacillus strains can be found in the intestinal microbiota of healthy humans, and the source of infection in those cases cannot be conclusively proven.49 In a review of the literature, there were no reports of bifidobacterial sepsis related to probiotic use; this is in keeping with animal studies that suggest low pathogenicity.50 Although Kunz et al51 described 2 premature infants who developed Lactobacillus bacteremia while taking Lactobacillus rhamosus GG (LGG) supplements, both of those preterm infants had short-gut syndrome; other authors and we did not observe sepsis attributable to probiotic organisms during the studies.15–18 The number of patients accumulated from all clinical trials may yield enough power to state that treatment is relatively safe, comparing the possible sepsis attributable to probiotics (0 of 940 patients; sum of all clinical trials including ours)17,18 with the higher incidence (7%–14%) and disastrous effects of NEC for VLBW infants.
The incidence of NEC or death was lower than the expected effect size we used in the sample size calculation. This may be partly a result of improvements in the quality of care for VLBW infants in Taiwan. It may also be attributable to the fact that the sickest infants were not enrolled in the study, because they either died before feeding or were not fed before 3 weeks of age. We conclude that probiotics containing B bifidum and L acidophilus, administered orally for 6 weeks, reduce the incidence of death or NEC for VLBW preterm infants.
This study was supported by the National Science Council of Taiwan (grant NSC 94-2314-B-039-007) and was approved by the institutional review board of China Medical University Hospital (proposal DMR94-IRB-14).
We thank our wonderful team members; this work would not have been possible without their active cooperation. We express sincere gratitude to Prof Wen-Miin Liang and Li-Na Liao in the Biostatistics Center, China Medical University, for their outstanding work on the statistical analyses. We also acknowledge the editorial assistance of Dr William Oh in the preparation of the manuscript.
- Accepted February 13, 2008.
- Address correspondence to Hung-Chih Lin, MD, Department of Pediatrics, China Medical University Hospital, Taichung, Taiwan, No. 2 Yu-Der Road, Taichung, 404, Taiwan. E-mail:
The authors have indicated they have no financial relationships relevant to this article to disclose.
Drs Lin and Hsu contributed equally to this report.
What's Known on This Subject
Orally administered probiotics reduce the incidence of NEC in VLBW preterm infants. However, optimal strains, timing, dosage, and duration of probiotic administration to VLBW preterm infants need to be further evaluated in large trials.
What This Study Adds
Bifidobacterium may be the most appropriate strain for the prevention of NEC. Probiotics containing Bifidobacterium bifidum and Lactobacillus acidophilus, administered orally for 6 weeks, reduced the incidence of death or NEC for VLBW preterm infants.
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- ↵Butel MJ, Roland N, Hibert A, et al. Clostridial pathogenicity in experimental necrotising enterocolitis in gnotobiotic quails and protective role of bifidobacteria. J Med Microbiol.1998;47 (5):391– 399
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