Necrotizing enterocolitis (NEC) and sepsis are leading causes of mortality and morbidity in premature infants; NEC has an incidence varying from 2,6 to 28% and an average mortality of 20–30%, up to 50% for infants requiring surgical treatment [
16]. Late onset sepsis (LOS) occurs approximately in 20% of very low birth weight (VLBW) infants and has a significant overall mortality risk and potential long-term neurodevelopmental sequelae [
17]. The onset of both conditions is rapidly progressive with involvement of multiple organs and treatment is unlikely to be wholly successful. Preventive strategies are urgently needed. The pathogenesis of NEC is multifactorial and incompletely understood, but the immaturity of intestinal barrier function and the development of an altered gut microbiota, with possible translocation of potentially pathogenic bacteria, may play an important role. Similar mechanisms are likely to be involved in cases of LOS when the infecting organism originates from a reservoir within the intestine [
18‐
20]. The importance of a diverse intestinal microbiome for good health in later life has been long recognized. These bacteria have a range of roles, improving the intestinal immunological defences, preventing colonization and possible invasion by pathogens through the intestinal wall. After preterm birth, acquisition of the microbiome is slow, typically less diverse, and dominated by
Enterobacteriaceae with relatively few
Lactobacilli and
Bifidobacteria. The contribution of abnormal patterns of the intestinal microbiota to the clinical onset of NEC and LOS is not clear, but there is evidence of perturbations of the gut microbiota in the period preceding the onset of clinical illness. The most recent Cochrane review of this topic, including 24 studies and more than 5500 infants, produced recommendations for the routine use of probiotics in prevention of NEC and mortality in preterm infants [
21]. However, concerns about the efficacy and safety of probiotics in this population have limited their introduction into clinical practice. Actually, a recent survey demonstrated that probiotic provision varies between 0 and 100% in 153 different NICU in the world [
22]. In the 5 years after the Cochrane review publication [
21], more clinical trials and metanalyses have been performed to better evaluate the role of probiotics in the prevention of NEC and LOS in preterm infants. In 2015 Aceti et al. on behalf of the Italian Society of Neonatology, performed a new systematic review: the results show an overall benefit of probiotic supplementation for the prevention of NEC [
23]. The 26 studies included in the metanalysis were extremely heterogeneous and in very few studies the same probiotic strain was used, weakening a strain specific sub-metanalysis. After pooling studies according to probiotic genus, no effect was documented for
Lactobacilli and
Saccharomyces; the analysis of studies using
Bifidobacteria showed a significant efficacy of
Bifidobacterium breve (B. breve) in reducing NEC [
23]. Both these findings are in contrast with the Cochrane review [
21]; the discrepancy may be due to differences in the studies included. Given the small number of trials reporting the rates of NEC in extremely low birth weight (ELBW) and intrauterine growth restriction (IUGR) infants, no specific recommendation was drawn in these two populations. In 2016, Costeloe et al. published a large RCT, the PiPs trial, recruiting more than 1000 babies [
24]. The authors chose a single strain probiotic product (
B. breve BBG-001) with the best available evidences at that time and an appropriate comparison group. No significantly differences were observed in LOS, NEC or death [
24]. The disappointing results of the PiPs trial caused a major drawback for the use of probiotics in the management of the preterms. In 2017, Uberos conducted a retrospective cohort study evaluating clinical outcomes in VLBW infants, before and after the introduction of routinely probiotics supplementation with
Lactobacillus rhamnosus GG (LGG) or Lactobacillus acidophilus (L. acidophilus) and Lactobacillus bifidum (L. bifidum) [
25]. The authors observed a significant reduction in NEC ≥ Stage II (11.3 vs 4.8%), LOS (16 vs 10.5%) and mortality (19.4 vs 2.3%) in infants born before 32 weeks of gestational age (WGA), while in neonates aged ≤27 weeks the reduction was not statistically significant [
25]. In the same year, Aceti et al., on behalf of the Italian Society of Neonatology published another metanalysis of 25 trials on probiotics and LOS: probiotics’ supplementation resulted in a significantly lower incidence of LOS (RR 0.79) [
26]. According to feeding type, the beneficial effect was confirmed only in exclusively human milk (HM) fed preterm infants, probably because of a synergic action of probiotics and prebiotics compounds of HM (RR = 0.75) and only for probiotic mixtures [
26]. Another review of 30 RCTs and 14 observational studies showed a reduction up to 50% of severe NEC and about 25% of all-cause mortality using of a two-probiotic combination
[L. acidophilus with Bifidobacterium infantis (B. infantis)] [
27]. Furthermore, a 12% reduction in the risk of sepsis in RCTs and a 19% reduction in observational studies were demonstrated. The metanalysis did not show a statistically significant effect in ELBW infants, because of insufficient number of clinical trials [
27]. In 2018 the ESPGHAN Working Group on Probiotics, Prebiotics and Committee on Nutrition published a detailed strain specific and network metanalysis (NMA) of data on probiotics, used in preterm infants [
28]. Differently from classical pair-wise meta-analyses which only address the comparative effectiveness among similar or competing interventions against a common comparator, NMA has the advantage to address multiple interventions simultaneously. Following this specific approach the authors were able to evaluate the efficacy of single probiotic strains or combination of studied strains [
28]. Fifty-one trials and over 11000 newborns were included. Most of the different probiotic strains were evaluated in one or two trials, while only 5 were studied in at least 4 RCTs. Noteworthy, the review included moderately preterm infants because trials focused on smallest babies were limited. The meta-analysis shows that only a minority of probiotic strains has a statistically significant effect in reducing mortality and morbidity in preterm infants. The absence of significant effects may reflect a lack of adequately powered RCTs, or a genuine lack of efficacy for those species or strains [
28]. The following probiotics were found in at least 2 trials to reduce: 1) NEC incidence:
Bifidobacterium lactis (B. lactis) Bb-12 or B94;
Lactobacillus reuteri (L. reuteri) ATCC 55730 or DSM 17938; LGG; the combination of
Bifidobacterium bifidum (B. bifidum),
B. infantis,
Bifidobacterium longum (B. longum) and
L. acidophilus; the combination of
B. infantis Bb-02,
B. lactis Bb-12, and
Streptococcus thermophilus (S. thermophilus) TH-4; the combination of
B. 35624 and
LGG; 2) LOS incidence: the combination of
B. bifidum,
B. infantis,
B. longum, and
L. acidophilus and the combination of
B. longum R00175,
Lactobacillus helveticus (L.helveticus) R0052,
L. rhamnosus R0011, and
Saccharomyces boulardii (S. boulardii) CNCM I-1079; 3) mortality: the combination of
B. bifidum NCDO 1453 and
L. acidophilus NCDO 1748. In 2019, a large metanalysis including 34 eligible studies and 9161 participants confirmed an advantage of probiotics in preventing NEC (3.54%) and gut-associated sepsis (15.59%), and in decreasing mortality (5.23%) in preterm infants [
29]. Probiotic mixtures showed the highest advantage. The same conclusion on LOS is reported by a previous review [
30]: pooled results from 37 RCTs and 9416 infants showed that probiotics significantly decreased the risk of LOS, with a number to treat of 44, in infants born < 37 WGA or < 2500 g. Beneficial effect in reducing LOS was reported by the analysis of studies including infants born < 32 WGA or < 1500 g. Subgroup analysis of extremely preterm infants (born < 28 WGA or < 1000 g) revealed no significant results. A recent single-centre retrospective observational study [
31] compared 2 different populations: 469 versus 513 preterm infants born before or after the introduction of routine daily supplementation of
Lactobacillus and
Bifidobacterium for prophylaxis of NEC. NEC rate fell from 7.5 to 3.1% after the introduction of this policy (~ 55% RR reduction). The improvement affected all high-risk neonates irrespective of gestation age and feeding type. LOS rates also fell from 22.6 to 11.5%. Moreover, all-cause mortality rate decreased, although this was consistent with a trend over the study period. The positive effect of probiotics appeared greatest in the first 2 weeks after birth, suggesting that early postnatal colonization is crucial. The authors supported routine use of multi-species
Lactobacillus and
Bifidobacterium combination for preventing NEC. A further review of 30 articles, with a total number of 9522 preterm infants involved, of which 4812 receiving probiotics, showed that the supplementation significantly reduces the incidence of stage II-III NEC (RR = 0.55) [
32]. Subgroup analysis indicated that mixed probiotics and
Lactobacillus reduce the incidence of NEC (for mixed probiotics, RR = 0.39; for
Lactobacillus RR = 0.53), while the individual use of
Bifidobacterium or
Saccharomyces did not have such effect. Similarly, probiotics’ supplementation significantly reduced the death rate (RR = 0.73), and subgroup analysis indicated that only mixed probiotics significantly reduce mortality (RR = 0.52), whereas
Lactobacillus,
Bifidobacterium, or
Saccharomyces alone did not reach the same result [
32]. More recently, after the last date of the search strategy of this review, ESPGHAN published an authoritative position paper on the use of probiotics in preterm infants [
33]. The authors provide a conditional recommendation (with low certainty of evidence) to use either
LGG or the combination of
B. infantis Bb-02,
B. lactis Bb-12, and
S. thermophilus TH-4 in order to reduce NEC rates [
33].