Necrotizing enterocolitis (NEC) is a gastrointestinal disease that seriously threatens the life of newborns. Clinically the infant presents with feed intolerance, increased gastric aspirates, vomiting, blood in the stool which may progress to very severe illness including shock and perforation. It is a disease that has plagued neonatal care for a long time and is still relatively common in very low birth weight infants (1). NEC is associated with neurodevelopmental delays, growth retardation, intestinal strictures and adhesions, and short bowel syndrome with or without intestinal failure (2). The high incidence of NEC cannot be ignored, and according to large multi-center neonatal network databases in the United States and Canada (3–5), NEC may occur in 7 out of 100 very low birth weight infants over the decades. Despite overall improvement in survival of preterm infants, a recent review suggests that the mortality and prevalence of NEC in very low birth weight infants has barely changed (6).

NEC, once established, is challenging to stop and has limited and expensive treatments. Treatment methods for NEC include antibiotics, gastric decompression, parenteral nutrition, etc. (7). It is not clear whether NEC is a single entity or a combination of similar entities and while progress has been made in understanding the pathogenesis of NEC there is still lack of clarity on many fronts which has perhaps contributed to a lack of significant progress in the treatment of NEC over the last many decades (8). On the other hand, although very low birth weight infants account for only a small proportion of newborns, the cost of treatment is indeed disproportionate. It has been reported that NEC causes more than 1 billion dollars in losses to medical institutions (9). It is worth noting that about 40% of NEC cases require surgical intervention (10), and the cost of treatment for infants requiring surgery has also significantly increased. All these factors lead to a considerable economic burden on the family and society.

Multiple research studies have explored various interventions for prevention of NEC including the provision of human milk and microbial therapeutics, with probiotic therapy garnering the most attention. Shiloh R. Lueschow et al. found that Bifidobacterium infantis EVC001 can prevent NEC in mice through anti-inflammatory and epithelial barrier-restoring properties (11). The study by Xiu-Li Zhu et al. has shown that probiotics supplementation can reduce the incidence and severity of NEC in preterm neonates (12), which seems to be related to the functions of probiotics in regulating immunity and inhibiting the imbalance of intestinal flora.

Most studies in the past decade have suggested that probiotics can significantly reduce the risk of NEC; However, it is unclear which probiotic or combination of probiotics is more effective (13) and what the optimal dose is. Moreover data on VLBW are scarce, and other related studies have not reported on specific strains of probiotics (14). Therefore, this study aimed to compare the effects of various probiotic regimens on NEC through a network meta-analysis, with direct or indirect comparisons, and to estimate the rank order of each combination. This hopefully will help target further research as well as facilitate improvements in practice.

NEC is a gastrointestinal disorder that has plagued the field of neonatology for a long time. Considering the morbidity and mortality of NEC, as well as the high cost of treatment and socioeconomic loss, it is important to prioritize research on NEC prevention and treatment. This study included 27 trials with 18 interventions, including 9,501 very low birth weight infants. We aimed to investigate which probiotics effectively prevent NEC in very low birth weight infants. This network meta-analysis concluded that Lactobacillus rhamnosus plus bovine lactoferrin might be the most appropriate regimen for preventing NEC in very low birth weight infants compared to a placebo or other probiotic control group.

Lactobacillus rhamnosus GG belongs to the genus Lactobacillus, a naturally occurring gram-positive bacterium that was originally isolated from the healthy human intestine (43). Lactobacillus rhamnosus GG has strong adhesion to intestinal cells and can also exert its high immune activity in the acidic pH environment of the digestive tract (44), which are prerequisites for colonization in the human intestine. Lactoferrin is a transferrin-like protein with anti-infective and anti-inflammatory properties (45) and is found in high levels in human colostrum and low levels in breast milk, tears, saliva, and semen. Lactoferrin can be processed from bovine or human milk, with GM rice and GM corn currently under study as promising new sources (46). Since bovine lactoferrin is cheaper than human lactoferrin, it is more readily used.

The studies by Paolo Manzoni et al. (15, 16) have shown that bovine lactoferrin combined with Lactobacillus rhamnosus GG can significantly reduce the incidence of NEC in very low birth weight infants, which is consistent with the results of this network meta-analysis. They believe that this might be related to the ability of lactoferrin to provide some anti-infection, nutrition, and immune regulation activity in the intestine to synergize with the effect of Lactobacillus rhamnosus GG against NEC in premature infants. These findings are also supported by a retrospective cohort study by Michael P. Meyer et al., who also showed that the cost of prevention was significantly lower than the cost of treatment (47). Lactobacillus rhamnosus GG can adhere to the intestinal epithelium and generate biofilms and attenuate the pro-inflammatory effects of cytokines and protect the mucosal barrier (48, 49). In addition, Lactobacillus rhamnosus GG can play an anti-pathogen role by stimulating non-specific immunity, increasing the secretion of interleukin-6 and expressing over 90 antimicrobial or immunomodulatory proteins (43, 48–50). Bovine lactoferrin may provide a broad-spectrum anti-pathogen effect by directly lysing microbial cell membranes (51, 52). Moreover, lactoferrin can also protect intestinal epithelial cells by down-regulating the highly expressed pro-inflammatory cytokines in intestinal epithelial cells, inhibiting the activity of free radicals and reducing the levels of oxidative products (53, 54).

It is obvious that Lactobacillus rhamnosus GG and bovine lactoferrin have the similar effects and create good conditions for the growth of beneficial bacteria, and can also inhibit the colonization of pathogens. The combined use can enhance the overall effect (55). The study by Po-Wen Chen et al. has shown that when the growth of probiotics is not optimum, bovine lactoferrin provides a more substantial prebiotic effect and promotes the growth of probiotics, including Lactobacillus rhamnosus GG (56).

A Position Paper by the European Society for Pediatric Gastroenterology Hepatology and Nutrition Committee on Nutrition and the European Society for Pediatric Gastroenterology Hepatology and Nutrition Working Group for Probiotics and Prebiotics indicates that the question of which probiotic strain or combination to use is mainly based on known literature (mainly case series and author's expertise) (57), these recommendations are based on very low certainty of evidence. Compared with other studies, this study compared the effects of various probiotic regimens on NEC through network meta-analysis, and obtained the optimal probiotic regimen by ranking each intervention. In addition, this study also analyzed specific strains of probiotics. In the studies included in this network meta-analysis, the use of probiotics is described as being well tolerated and safe in very low birth weight infants. Feeding intolerance and clinical sepsis were significantly reduced in the probiotic group compared to the control group. Interestingly, these studies also suggest that different outcomes may be influenced by feeding type (human milk vs. formula). This appears to further support the benefits of lactoferrin in combination with probiotics. Combining probiotics and lactoferrin may be a good idea for future research studies.

This network meta-analysis also has some limitations. This study only discussed the selection of probiotics for the prevention of NEC in very low birth weight infants, while the questions of the dosage, the timing of the intervention, and when to start the intervention remains unresolved. Most interventions were evaluated in only one or two trials, and only a few options were tested in four randomized controlled trials. Therefore, most probiotic interventions were evaluated in small experimental populations. In conclusion, the results of this study should be interpreted with caution, as the number of included trials was insufficient, so there was limited evidence for direct comparisons of some interventions, and further related studies are needed.

Our analysis suggests that Lactobacillus rhamnosus GG combined with bovine lactoferrin is the most effective and recommended regimen for preventing NEC in very low birth weight infants. However further studies are required to confirm this and also answer questions about probiotic dosage, timing and duration of therapy.