Necrotising enterocolitis (NEC) is one of the most common and serious acquired bowel diseases a newborn can face. Although it is not fully understood, the cause is suspected to be a combination of vascular necrosis, bacterial overgrowth and metabolic insults to the immature gut wall. It is believed that after toxic or ischaemic damage to the mural wall, and using the substrate proceeding from enteral feedings, there is an overgrowth of bacteria, especially those generating gases such as methane or hydrogen, producing intramural gas (pneumatosis intestinalis) leading to a mural necrosis, gut gangrene and, finally, intestinal perforation with peritonitis. In recent years, there has also been a greater acceptance of the theory that the host's immune system also plays an important pathophysiological role in the development of NEC.1

NEC predominantly affects premature infants, with 70–85% of cases of NEC occurring in very low birth weight infants (<1500g) or infants younger than 32 weeks, whereas only 10–25% of cases are term infants or late preterm infants. The lower the gestational age, the higher the risk of NEC. Overall incidence of NEC has been reported as 7% in a study conducted by the National Institute of Child Health (NICHD) Neonatal Research Network (NRN) in a population of 11,072 very low birth weight infants (VLBW) for the period 1999–2001.2 Another study, between the years 2003 and 2007, found that the prevalence of NEC was about 11% in very premature infants who were born at between 22 and 28 weeks post-menstrual age and had birth weights of 401–1500g.3 The global mortality of NEC is approximately 20–40%, rising to 90–100% in the most severe cases, which represents 2–5% of all neonatal intensive care unit (NICU) admissions.4 In addition, between 27% and 63% of affected infants will undergo surgery5 and one third will have a perforated bowel, the short bowel syndrome being the most common complication.

Probiotics can be defined as live microorganisms which, when administered in adequate amounts, confer a health benefit for the host (WHO/FAO 2001). Members of the genera Lactobacillus and Bifidobacterium are the most commonly used, the Lactobacillus rhamnosus GG and Bifidobacterium Lactis being the strains with stronger evidence to support their use in humans. Although the origin of the human microbiota is yet unknown, it is important to note the differences after birth depend on the method of delivery, type of feeding during gestation, as well as the mother's microbiota.6 Healthy breast-fed infants have a higher amount of bifidobacteria whereas formula-fed infants tend to have a more varied range of microorganisms such as bifidobacteria, bacteroidetes, enterobacteria, streptococci and clostridia. The human microbiota will not be stable until the age of 2–3 years and, even then, might suffer modifications due to diet, disease, antibiotic use and ageing. In recent years, probiotics have been increasingly studied and used in clinical practice. Some examples of this are the improvement of intestinal function in cystic fibrosis, reducing bacterial overgrowth,7 or the shortening of the duration of acute gastroenteritis.8

VLBW infants with increased risk of NEC have different faecal bacteria colonisation, together with delayed onset of bacterial colonisation in comparison to normal term newborns.9 It is believed that probiotics may modify the microbiota and the immune response of the newborn, reducing the risk of NEC through different mechanisms; direct competition for nutrients and prebiotics, transforming certain elements present in the gut into inhibitory substances, producing growth factors and vitamins to promote a healthy microbiota, synthesising bacteriocins, competing for binding locus, increasing the production of IgA, increasing the immune response and reducing the inflammation when stimulating regulatory lymphocytes through IL-10 and TGFβ.10–12 Despite the potential benefits, such interventions have been delayed due to the theoretical risk of bacterial sepsis that could occur in VLBW or preterm infants, especially those with extremely low birth weight (<1000g). However, there is little data to support this concern. In addition, infants exposed to a NICU undergo a wide range of procedures that may modify their microbiota: antibiotic use, diet modifications, catheterisation, etc.

The use of probiotics has been analysed and studied by different scientific societies. An example would be the Spanish Society of Neonatology (SENeo), which provides the following reccommendations13:

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  In situations with high NEC incidence, it is recommended to provide probiotic supplementation. Grade 1+.

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  Probiotic use should be considered in neonates under 32 weeks or less than 1500g, including less than 1000g newborns. Good ethics practice.

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  Probiotic use requires a strict monitoring. Good ethics practice.

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  Evidence suggests the use of species from the Lactobacillus and Bifidobacterium genres, due to their effect in reducing NEC, although the species, dose and duration must be adapted according to each case. Good ethics practice.

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  Start the administration as soon as possible and maintain it 6 weeks or until discharge. Good ethics practice.

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  Stop probiotic supplementation in case of NEC or gastrointestinal disease. Good ethics practice.

Up to the present, there have been few studies comparing the effects of specific strands of probiotics, alone or in combination, on NEC, late-onset sepsis and mortality. The objective of this study is to compare different probiotic mixtures to prevent necrotising enterocolitis and reduce late-onset sepsis and mortality in premature infants under 1500g.

A total of 24 publications were selected to undergo further study, of which 9 were selected for their high quality and reduced risk of bias (Fig. 1). It is important to note that trials were highly variable according to their inclusion criteria, risk of NEC, sample size, as well as the dose, the type and the duration of probiotics given. The characteristics of these trials are summarised in Table 2.

Figure 1.

Flux diagram of study selection.

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Effect of probiotics on severe NEC (stage II–III)

Data on severe NEC were present in all 9 studies. There were a total of 3521 newborn infants divided into two groups: the probiotic group and the control group. In the probiotic group, there were a total of 1756 neonates whereas the control group had a total of 1765 infants. There was a statistically significant reduction in the relative risk of developing NEC in the probiotic group in comparison to the control group, RR 0.39; 95% CI: 0.26–0.57. The newborns needed to treat with probiotics to prevent 1 case of NEC were 33; 95% CI: 23–54 (Table 3).

Table 3.

Probiotics vs control, severe NEC (stage II-III) in <1500g infants.

Studies	NEC probiotic	NEC control	Relative Risk
Dani et al. (2002)	4/295	8/290	0.49 [0.15–1.61]
Lin et al. (2005)	2/180	10/187	0.21 [0.05–0.94]
Lin et al. (2008)	4/217	14/217	0.29 [0.10–0.85]
Manzoni et al. (2009)	0/151	3/153	0.05 [0.00–0.90]
Rougé et al. (2009)	2/45	1/49	2.18 [0.2–23.21]
Samanta et al. (2009)	5/91	15/95	0.34 [0.13–0.92]
Sari et al. (2010)	6/110	9/111	0.12 [0.01–2.19]
Braga et al. (2011)	0/119	4/112	0.10 [0.01–1.92]
Jacobs et al. (2013)	11/548	24/551	0.56 [0.31–0.99]
Global	34/1756	88/1765	0.39 [0.26–0.57]
NNT	33 [23–54]

In order to study if the probiotic strand had any influence on NEC reduction, data from the Dani 2002 and Manzoni 2009 vs the Lin 2005 and Lin 2008 vs Samanta 2009 and Jacobs 2013 were analysed. In the first two, L. rhamnosus GG (Bivos®) was used, whilst in the following two, they used Lactobacillus acidophilus with Bifidobacterium bifidum (Infloran®). Finally, in the last two studies, three or more probiotics were used (Bifidobacterium infantis, Streptococcus termophilus, B. lactis, B. bifidum, Bifidobacterium longum and L. acidophilus).

When studying the three different groups, 446 newborns were treated with Bivos®, 397 with Infloran® and 639 neonates with 3 or more probiotics. The 1st group of only 1 probiotic showed no statistically significant reduction in NEC incidence, whereas the 2nd of 2 probiotics and the 3rd of 3 or more probiotics did have a significant reduction in relative risk (Table 4). As for the NNT, in the 2nd group the NNT was the lowest of the three, NNT 22; 95% CI: 14–54, followed by the 3rd group, NNT 28; 95% CI: 17–75. Once summarised to the same number of cases, and after applying chi-square analysis, there was no statistically significant difference between them (Fig. 2).

Table 4.

Probiotic strand associated differences.

	Probiotic	Control	Relative risk	NNT
NEC (stage II-III)
1 probiotic (Bivos®)	4/446	11/443	0.36 [0.12–1.13]	–
2 Probiotics (Infloran®)	6/397	24/404	0.26 [0.11–0.62]	22 [14–54]
≥3 probiotics	16/639	39/646	0.41 [0.23–0.73]	28 [17–75]
Mortality
1 probiotic (Bivos®)	6/446	6/443	0.99 [0.32–3.06]	–
2 Probiotics (Infloran®)	9/397	29/404	0.32 [0.15–0.66]	20 [12–50]
≥3 probiotics	31/639	42/646	0.74 [0.47–1.17]	–
Sepsis
1 probiotic (Bivos®)	21/446	21/443	0.99 [0.55–1.79]	–
2 Probiotics (Infloran®)	62/397	60/404	1.05 [0.76–1.46]	–
≥3 probiotics	85/639	117/646	0.73 [0.57–0.95]	21 [11–119]

Figure 2.

Graphs.

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Although there have been systematic publications and good quality reviews that sustain the correct use of probiotics for preventing NEC, probiotics have still not truly been implemented worldwide. In order to provide more data supporting the use of probiotics as a medical tool to prevent NEC, this meta-analysis was performed using only high-quality studies, based on their allocation concealments procedures and blinding of the intervention. Therefore, after making a pre-selection of 24 studies, only 9 of them where finally considered adequate for the analysis given that they had a good allocation concealment procedure, good blinding interventions and also provided information on all factors that influence the incidence of NEC.

Regarding the study selection criteria, infants less than 34 weeks or weighing less than 1500g were selected due to their increased risk of suffering from NEC. In addition, the majority of studies also contemplated these age/weight groups for the aforementioned reasons. Moreover, preterm infants will experiment a growth in number, especially in those countries where maternal age increases and with the upsurge in artificial methods of conception. Under such circumstances, it seems important to pay special attention to the group with a higher risk of NEC for which a prevention method could be very important. In term infants, or late preterm infants, the incidence of NEC is significantly lower, meaning that the costs of preventing a NEC case could be much higher than the possible risks associated with NEC. Furthermore, the use of probiotics for prevention could render no positive results. For the bias risk assessment, the Jadad score was used because it has been recognised as a good method for reducing the blinding bias, in conjunction with others, and increases the statistical quality of systematic reviews and meta-analysis.24 In addition to the use of the Jadad score, the standard methods of the Cochrane Collaboration and Neonatal Review Group were also taken into account since this institution is very well regarded for its scientific and research rigour.25

When considering the actual effect of probiotics in NEC prevention, it is clear that there is a reduction of severe NEC, consistent with many other existing meta-analyses5,26–31 and studies, as well as a reduction in mortality without any effect on late-onset sepsis: neither reduction nor increase (late-onset sepsis is the principal feared risk of giving such prophylactic measures). Although the numbers needed to treat to prevent a NEC case are relatively high, 33, and the numbers to prevent death in neonates are even higher, 53, these have to be taken into account when facing the fact that premature births are increasing, the costs of a NEC episode are high and probiotics are easy to handle in addition to having a low cost. However, since NEC has a high morbimortality, would it not be unethical to keep this inexpensive measure from newborns? In future trials, it would seem better to focus on the effect of different probiotic strands in order to identify the best combination possible for these patients. Following this direction, in the current meta-analysis, strand-associated difference was studied and noted. Although there was no difference between probiotics when it came to reducing NEC incidence, there was a clear benefit in death reduction when giving a two probiotic combination (L. acidophilus and B. bifidum), compared to three or more probiotics (B. infantis, S. termophilus, B. lactis, B. bifidum, B. longum and L. acidophilus) or just one probiotic (L. rhamnosus GG). When focusing on late-onset-sepsis, the combination of 3 or more probiotics proved to be beneficial when compared to the rest. Therefore, both the combination of 2 probiotics and 3 or more probiotics would be beneficial for the newborns, one especially for mortality and the other for late-onset sepsis.

As for the dose of probiotics, it is important to achieve an optimal mass of probiotic in order to survive and colonise the gut, proliferating in adequate amounts, to confer a health benefit. The evidence suggests this dose should be minimally 106–107 colony-forming units in each gram of product.32–34

Finally, concerning the strengths and weaknesses, this meta-analysis is the first to study the different probiotic compounds and mixtures existing and the first to find out if there is a probiotic mix or compound with superior performance when compared with the rest. However, neither the mix of probiotics nor the dose nor the duration of their administration are always the same, leading to a reduction in the certainty of the results. Moreover, in some cases, although concealment and blinding of intervention were correct, there was a substantial difference in the age at birth between groups, a fundamental factor for the risk of NEC, making it difficult to know if the reduction of relative risk was due to probiotics or simply because the maturity was different between groups. Other studies, although still correct, had poor data analysis and lacked information concerning the kind of feeding infants received, or lacked information about some risk or prevention factors concerning NEC. It is also important to note that no studies have assessed the effect of probiotics in extremely low birth weight neonates (<1000g) where the risk of late-onset sepsis could be higher.

To conclude, the use of probiotics as a method to prevent severe necrotising enterocolitis in preterm neonates has been proven, with the combination of L. acidophilus and B. bifidum emerging as the best in reducing overall cause mortality. Therefore, probiotics should be given routinely to all high-risk neonates. Where there are safety concerns, probiotics could be continued within a research framework that does not necessarily need studies with a placebo comparison.

Authors declare no conflict of interest.