Acta Pædiatrica ISSN 0803-5253

REVIEW ARTICLE

Probiotics for the prevention of necrotising enterocolitis in very low-birth-

weight infants: a meta-analysis and systematic review
John P. Thomas (drjohnpthomas@gmail.com)1, Tim Raine2, Sanath Reddy3, Gusztav Belteki4
1.Department of Gastroenterology, Norfolk and Norwich University Hospital, Norwich, UK
2.Division of Gastroenterology, Department of Medicine, Addenbrooke’s Hospital, University of Cambridge, Cambridge, UK
3.Department of Paediatrics, Princess Alexandra Hospital, Harlow, UK
4.Department of Neonatology, Cambridge University Hospitals NHS Trust, Cambridge, UK

Keywords ABSTRACT
Necrotising enterocolitis, Nutrition, Probiotics, Very We performed an updated meta-analysis incorporating the results of recent randomised
low-birth-weight infant
controlled trials (RCTs) to measure the effectiveness of probiotic supplementation in
Correspondence preventing necrotising enterocolitis (NEC) and death in very low-birth-weight (VLBW)
Dr John P. Thomas, Norfolk and Norwich University
Hospital, Colney Lane, Norwich, NR4 7UY, UK.
infants, and to investigate any differences in efficacy by probiotic agent. Using meta-
Tel: 01603 288358 | regression analysis, we assessed the contribution of other measured variables on the
Fax: 01603 288368 | overall effect size and between-study variability.
Email: drjohnpthomas@gmail.com
Conclusion: Overall, probiotics lead to significant reductions in NEC incidence and
Received mortality in VLBW infants. Differences in probiotic agents and the influence of prenatal
6 February 2017; revised 25 March 2017;
accepted 28 April 2017.
steroids and feeding regimens may explain the differences in outcomes between studies.

DOI:10.1111/apa.13902

INTRODUCTION largely unchanged since it was first characterised by

Necrotising enterocolitis (NEC) is one of the leading causes
of neonatal mortality and morbidity in very low-birth-
weight (VLBW) infants (1). It is the most common
gastrointestinal neonatal emergency (2), with a prevalence
of up to 10% and a mortality of 20–30% in developed
countries (3). NEC is also associated with long-term
complications including neurodevelopmental delay and
short-gut syndrome (1). Despite extensive research, the
incidence, morbidity and mortality of NEC have remained
Abbreviations
b.d, Twice daily; BBi, Bifidobacterium bifidum; BBr, Bifidobac-
terium breve; BI, Bifidobacterium infantis; BLa, Bifidobacterium
lactis; BLo, Bifidobacterium longum; BM, Breastmilk; c.f.u,
Colony forming units; CI, Confident intervals; ELBW, Extremely
low-birth-weight; FDA, Food and Drugs Agency; FFF, From first
enteral feed; FM, Formula milk; GA, Gestational age; GB,
Gusztav Belteki; IL-1, Interleukin-1; IL-8, Interleukin-8; JT, John
Thomas; LA, Lactobacillus acidophilus; LC, Lactobacillus casei;
LP, Lactobacillus plantarum; LPS, Lipopolysaccharide; LRe,
Lactobacillus reuteri; LRh, Lactobacillus rhamnosus; LS, Lacto-
bacillus sporogenes; MHRA, Medicines and Health products
Regulatory Agency; NEC, Necrotising enterocolitis; NS, Not
stated in original study; o.d, Once daily; PMA, Postmenstrual age;
PRISMA, Preferred Reporting Items for Systematic Reviews and
Meta-Analyses; PS, Probiotic supplementation; q.d.s, Four times
daily; RCTs, Randomised Controlled Trials; RR, Relative Risk;
SB, Saccharomyces boulardii; SR, Sanath Reddy; ST, Streptococ-
cus thermophilus; TR, Tim Raine; UK, United Kingdom; USA,
United States of America; VLBW, Very low-birth-weight.
Schmidt and Quaiser in 1953 (4).
The underlying problem in NEC is believed to be a
pathologic interaction between an immature gastrointesti-
nal system and microbial agents colonising the preterm gut
and chemical substances from food. Recent research has
revealed that intestinal dysbiosis, coupled with poor intesti-
nal barrier function and an over-reactive immune response
involving pattern recognition receptor pathways, may be
the principle mechanisms by which NEC arises (5,6).
However, the precise pathogenesis and the interplay
between these mechanisms are far from being fully
elucidated.
Several clinical trials, especially in the past decade, have
assessed the utility of agents designed to manipulate these
Key notes
 This updated meta-analysis demonstrates that probiotic
supplementation in very low-birth-weight infants leads
to statistically significant reductions in the incidence of
necrotising enterocolitis (NEC) stage ≥2 and all-cause
mortality.
 Subgroup analysis identified formulations containing
Bifidobacterium and Lactobacillus species to be the
most efficacious.
 Meta-regression analysis revealed that probiotics were
most effective in cohorts with higher levels of exclusive
breastmilk feeding and lower levels of prenatal steroid
exposure.

©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd 1
Probiotics for preventing NEC in VLBW infants
key pathways in the hope of preventing NEC. Probiotics,
prenatal glucocorticoids, breastmilk, standardised feeding
protocols and bovine lactoferrin have been shown to reduce
the risk of NEC in systematic reviews (7). Of these,
probiotics have attracted great attention but also contro-
versy owing to varying reports regarding their efficacy and
safety. Probiotics are defined by the World Health Organ-
isation as ‘live micro-organisms which, when administered
in adequate amounts, confer a health benefit on the host’
(8). Although randomised controlled trials (RCTs) have
shown conflicting results, several meta-analyses have con-
sistently demonstrated a statistically significant beneficial
effect of probiotics in preventing NEC and reducing all-
cause mortality (9–14). This has led some experts to
advocate the routine introduction of probiotics across all
neonatal units (15). However, there are still many unan-
swered questions including the optimal probiotic agent, the
dose and duration of supplementation, the impact of
potential treatment effect modifiers such as breastfeeding,
as well as short-term and long-term adverse effects (16,17).
We have performed a systematic review and updated
meta-analysis, in compliance with PRISMA guidelines, to
evaluate the efficacy of probiotic supplementation in pre-
venting NEC and death in VLBW infants, and to investigate
any differences in efficacy by probiotic agent. We have
utilised meta-regression to test for underlying associations
that may explain the differences seen in outcomes and
hence to suggest future study designs to investigate the
potential beneficial role for probiotics in NEC.
METHODS
Literature search
A literature search was performed for studies published
before 1st August 2016 in MEDLINE (via PubMed),
EMBASE and the Cochrane Library databases. The full
search string conducted in PubMed is shown in Figure 1.
Previous meta-analyses on this subject were also screened
to identify additional relevant studies.
Selection of studies
Only studies fulfilling a number of criteria were selected for
further analysis: (i) RCTs of a probiotic regimen versus
placebo or nonplacebo control; (ii) Study populations
involving VLBW infants (i.e. neonates with a birthweight
of <1500 g); (iii) Administration of probiotic agent within
two weeks of birth; (iv) Primary or secondary outcomes of
NEC stage ≥2 [according to Bell’s staging criteria (18)] and
mortality; (v) Papers written in English.
Assessment of methodological quality
Key characteristics of each study were evaluated indepen-
dently by two authors (JT and SR) including population
size, study period, intervention methodology, randomisa-
tion technique, blinding strategy and differences in treat-
ment effect modifiers. All studies were independently
scored against the Jadad scale to quantitatively assess the
methodological quality. After comparison of these scores,
2 ©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd
Thomas et al.
any differences were reconciled following discussions with
TR and GB. A minimum Jadad score of 3 had to be achieved
for a study to be included in the analysis.
Statistical analysis
A meta-analysis was used to measure the association
between probiotic supplementation and incidence of NEC
stage ≥2 [defined according to Bell’s criteria (18)],
all-cause mortality (death from any cause) and NEC-
related mortality (death as a result of NEC) in VLBW
infants. Separate meta-analyses were also carried out to
assess the efficacy of probiotics according to the study
location and in the extremely low-birth-weight (ELBW)
infant cohort (i.e. neonates with a birthweight of
<1000 g). A meta-analysis of subgroups was performed
out to assess for differences in outcomes according to the
probiotic agent.
Due to underlying differences in the recruited study
populations and intervention strategies, we used random-
effects models rather than a fixed-effect approach. Hetero-
geneity was estimated using the Cochrane’s Q and I2
statistics (19). Risk ratios (RR) and 95% confidence inter-
vals (CI) across multiple studies were calculated using the
Mantel–Haenzel method (20). The test statistic of overall
intervention effect, Z, was computed to attain the signifi-
cance of the result. Forrest plots were used to illustrate the
results of the meta-analyses. Funnel plots and the Egger’s
regression test(19) were used to assess publication bias in
analyses involving at least 10 RCTs. Multivariate meta-
regression analysis was performed to assess the impact of
treatment effect modifiers on the overall effect (20).
A two-tailed p-value of less than 0.05 was defined as
statistically significant for all analyses apart from
Cochrane’s Q test for heterogeneity where a p-value of
0.10 was selected as the threshold of significance.
The main meta-analysis and subgroup analysis were
performed using Review Manager 5.3 (Cochrane Collabo-
ration). Publication bias and meta-regression analysis were
computed using Comprehensive Meta-analysis Version 3
(Biostat USA).
RESULTS
Literature search
The literature search in MEDLINE/PubMed identified 324
studies. No additional studies were found in the other
databases. Of the 324 studies, only 37 were identified as
RCTs. Two further RCTs were identified after reviewing
previous meta-analyses. All identified RCTs were then
assessed for eligibility for inclusion in the analysis, after
which 15 RCTs were excluded (Fig. 1).
Study methodological quality
The Jadad scores are shown in Table 1 (also Table S1). Only
one study [Kitajima et al. (21)] failed to achieve a score of
3/5 due to a lack of adequate blinding and randomisation.
As a result, this study was also excluded, leaving 23 RCTs
remaining in the analysis.
Thomas et al.
Identification
Screening
Eligibility
Included
Figure 1 PRISMA Flow chart.
Study characteristics
The key study characteristics and primary outcomes of the
23 RCTs are summarised in Table 1.
The clinical and demographical details of the infants who
received probiotics are in Table S2. Probiotic formulation
and supplementation are presented in Table S3. NEC stage
≥2 and all-cause mortality in VLBW infants were outcome
measures present in 23 and 22 trials, respectively. NEC-
related mortality was only assessed by 10 studies.
Probiotics and incidence of NEC stage ≥2 in VLBW
infants
There were a total of 7325 neonates across all 23 RCTs,
comprising of 3703 neonates in the probiotic group and
3622 neonates in the control group (Fig. 2A). Fewer infants
in the probiotic group (145 (3.9%) infants) developed NEC
stage ≥2 in comparison with the control group [240 (6.6%)].
This difference was statistically significant with a RR of 0.57
(95% CI: 0.43–0.74, p < 0.0001). The Cochrane Q test
(26.78, p = 0.18) and I2 statistic (22%) revealed that the
degree of heterogeneity was not statistically significant
(p > 0.10).
©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd
Probiotics for preventing NEC in VLBW infants
Probiotics and mortality in VLBW infants
Twenty-two RCTs reported data for all-cause mortality.
Fewer deaths occurred in the probiotic group [174/3527
(4.9%)] compared with the control group [232/3427
(6.8%)] (Fig. 2B). This reduction in all-cause mortality
was statistically significant with a RR of 0.72 (95% CI: 0.57–
0.92, p = 0.009). The degree of heterogeneity was not
statistically significant (Cochrane’s Q = 22.88, p = 0.24).
NEC-related mortality data were also compared between
probiotic and control groups, in trials where this data were
available (Fig. 2C). Although fewer NEC-related deaths
occurred in the probiotic group [24/2318 (1.0%)] compared
with the control group [39/2324 (1.7%)], this failed to
achieve statistical significance (RR = 0.64, 95% CI: 0.38–
1.07, p = 0.09). There was no statistically significant hetero-
geneity (Cochrane’s Q = 5.03, p = 0.75).
Probiotics in ELBW infants
Only five RCTs reported data for NEC incidence in the
ELBW infant population. Fewer ELBW infants in the
probiotic group (71 of 797 [8.9%]) group developed NEC
stage ≥2 compared to the control group (85 of 799 [10.6%]),
3
 4
Table 1 Key study characteristics and outcomes
NEC-related NEC-related
NEC stage ≥2 in NEC stage ≥ 2 All-cause mortality All cause mortality mortality in mortality in
Number of Jadad Probiotic probiotic group, in control Relative Risk, in control group, in probiotic Relative Risk control group, probiotic Relative
Study Location centres Score composition (%) group, (%) (95% CI) (%) group, (%) (95% CI) (%) group, (%) Risk (95% CI)

Costeloe (22) England 24 5 BBr 61/650, (9.4%) 66/660, (10%) 0.93 (0.68–1.27) 56/660, (8.5%) 54/650, (8.3%) 0.93 (0.67–1.30) 14/660, 9/650, 0.62
(BBG-001) (2.1%) (1.4%) (0.21–1.84)
Dilli (23) Turkey 1 5 BLa 2/100, (2%) 18/100, (18%) 0.11 (0.03–0.47) 12/100, (12%) 3/100, (3%) 0.25 (0.07–0.86) NS NS NS
Hays (24) France 3 5 BLa, BLo 8/145, (5.5%) 3/52, (5.8%) 0.96 (0.26–3.47) 5/145, (12%) 1/52, (3%) 1.79 (0.21–14.99) NS NS NS
Totsu (25) Japan 19 4 BBi 0/153, (0%) 0/130, (0%) Not estimable 2/153, (1.3%) 0/130, (0%) 4.25 (0.21–87.8) NS NS NS
Patole (26) Australia 1 5 BBr (M-16V) 0/77, (0%) 1/76, (1.3%) 0.33 (0.01–7.95) 0/77, (0%) 0/76, (0%) Not estimable NS NS NS
Oncel (27) Turkey 1 4 LRe 8/200, (4%) 10/200, (5%) 0.80 (0.32–1.99) 15/200, (7.5%) 15/200, (7.5%) 0.75 (0.40–1.42) 4/200, 3/200, 0.75
(2%) (1.5%) (0.17–3.31)
Probiotics for preventing NEC in VLBW infants

Saengtawesin Thailand 1 3 BBi, LA 1/31, (3.2%) 1/29, (3.4%) 0.94 (0.06–14.27) 0/31, (0%) 0/29, (0%) Not estimable 0/31, 0/29, Not
(28) (0%) (0%) estimable
Jacobs (29) Australia 10 5 BI, ST, BLa 11/548, (2%) 24/551, (4.4%) 0.46 (0.23–0.93) 28/551, (5.1%) 27/548, (4.9%) 0.97 (0.58–1.62) 11/551, 4/548, 0.37
(2.0%) (0.9%) (0.12–1.14)
Demirel (30) Turkey 1 5 SB 6/135, (4.4%) 7/136, (5.1%) 0.86 (0.30–2.50) 5/136, (3.7%) 5/135, (3.7%) 1.01 (0.30–3.40) NS NS NS
Serce (31) Turkey 1 5 SB 7/104, (6.7%) 7/104, (6.7%) 1.00 (0.58–1.72) 4/104, (3.8%) 5/104, (4.8%) 1.25 (0.35–4.52) 3/104, 3/104, 1.00
(2.9%) (2.9%) (0.21–4.84)
Fernandez- Mexico 1 5 LA, LRh, 6/75, (8%) 12/75, (16%) 0.54 (0.21–1.39) 7/75, (9.3%) 1/75, (1.3%) 0.14 (0.01–1.13) NS NS NS
Carrocera (32) LC, LP, BI,
ST
Al-Hosni (33) US 3 3 LRh GG, BI 2/50 (4%) 2/51 (3.9%) 1.02 (0.15–6.96) 4/50, (8%) 3/50, (6%) 0.77 (0.18–3.25) NS NS NS
Rojas (34) Columbia 9 4 LRe 6/176 (3.4%) 10/194 (5.2%) 0.66 (0.25–1.78) NS NS NS NS NS NS
Sari (35) Turkey 1 5 LS 6/110, (5.5%) 10/111, (9%) 0.61 (0.23–1.61) 4/111, (3.6%) 3/110, (2.7%) 0.76 (0.17–3.30) 1/111, 0/110, 0.34
(0.9%) (0%) (0.01–8.17)
Braga (36) Brazil 7 5 LC, BBr 0/119, (0%) 4/112, (3.6%) 0.10 (0.01–0.77) 27/112, (24.1%) 26/119, (21.8%) 0.91 (0.56–1.45) NS NS NS
Mihatsch (37) Germany 1 3 BLa (BB12) 2/91, (2%) 4/89, (4%) 0.49 (0.09–2.60) 1/89, (1%) 2/91, (2%) 1.96 (0.18–21.19) 0/89, 1/91, 2.93
(0%) (1%) (0.12, 71.10)
Rouge (38) Spain 2 4 LRh GG, BLo 2/45 (4.4%) 1/49 (2.0%) 2.18 (0.20–23.21) 2/45, (4.4%) 4/49, (8.2%) 0.54 (0.10–2.83) NS NS NS
(BB536)
Samanta (39) India 1 3 BI, BBi, BLo, 5/91, (5.5%) 15/95, (15.8%) 0.35 (0.13–0.92) 14/95, (14.7%) 4/91, (4.4%) 0.30 (0.10–0.87) NS NS NS
LA
Lin (40) Taiwan 7 5 BBi, LA 4/217, (1.8%) 14/217, (6.4%) 0.29 (0.10–0.85) 9/217, (4.1%) 2/217, (0.9%) 0.22 (0.05–1.02) 3/217, 2/217, 0.67
(1.4%) (0.9%) (0.11–3.95)
Manzoni (41) Italy 1 5 LRh 1/39, (2.5%) 3/41, (7.5%) 0.35 (0.04–3.23) 6/41, (15.4%) 5/39, (12.5%) 0.88 (0.29–2.64) NS NS NS
Bin-Nun (42) Israel 1 4 BBi, BI, ST 1/72, (1%) 10/73, (14%) 0.10 (0.01–0.77) 8/73, (11.0%) 3/72, (4.2%) 0.38 (0.11–1.38) 3/73, 0/72, 0.14
(4.1%) (0%) (0.01–2.75)
Lin (43) China 1 5 LA, BI 2/180, (1.1%) 10/187, (5.3%) 0.21 (0.05–0.94) 20/187, (10.7%) 7/180, (3.9%) 0.36 (0.16–0.84) NS NS NS
Dani (44) Italy 12 4 LRh 4/295, (1.4%) 8/290, (2.8%) 0.49 (0.15–1.61) 2/290, (0.7%) 0/295, (0.0%) 0.20 (0.01–4.08) 0/290, 2/295, 4.92
(0%) (0.7%) (0.24–101.95)

BM = Breastmilk, FM = Formula milk, BM/FM = BM &/or FM, PS = Probiotic supplementation, o.d = Once daily, b.d = Twice daily, q.d.s = Four times daily, c.f.u = Colony forming units, FFF = From first enteral
feed, PMA = Post-menstrual age, GA = Gestational age, NS = Not stated in original study.
Probiotic types: LA = Lactobacillus acidophilus, BLa = Bifidobacterium lactis, BI = Bifidobacterium infantis, BBr = Bifidobacterium breve, BBi = Bifidobacterium bifidum, BLo = Bifidobacterium longum,
LRh = Lactobacillus rhamnosus, ST = Streptococcus thermophilus, LRe = Lactobacillus reuteri, LC = Lactobacillus casei, LP = Lactobacillus plantarum, LS = Lactobacillus sporogenes, SB = Saccharomyces
boulardii.

©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd
Thomas et al.
Thomas et al. Probiotics for preventing NEC in VLBW infants

A NEC stage ≥ 2

B All-cause deaths

C NEC-related deaths

Figure 2 Forest plot comparing probiotics vs control in VLBW infants, outcomes: (A) NEC stage ≥2 (B) All-cause deaths C) NEC-related deaths.

©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd 5
Probiotics for preventing NEC in VLBW infants
A NEC stage ≥ 2
B All-cause deaths
Figure 3 Forest plot comparing probiotics vs control in ELBW infants, outcomes: (A) NEC stage ≥2 (B) All-cause deaths.
but this was not statistically significant with a RR of 0.86
(95% CI: 0.65–1.16, p = 0.32; Fig. 3A).
Four RCTs reported data for all-cause mortality in the
ELBW infant population. Fewer deaths (60 of 562 [8.9%])
occurred in the probiotic group compared with the control
group (80 of 559 [14.3%]), but this was also not statistically
significant with a RR of 0.78 (95% CI: 0.50–1.20, p = 0.26;
Fig. 3B).
Probiotics and study location
A meta-analysis was also performed to evaluate any differ-
ences in efficacy of probiotics according to the study
location (Fig. 4). Only studies located in Asia and Australia
demonstrated statistically significant reductions in NEC
stage ≥2 incidence with a RR of 0.31 (95% CI: 0.17–0.59,
p = 0.0003) and RR of 0.45 (95% CI: 0.23–0.90, p = 0.02),
respectively. Studies performed in the Middle East (RR =
0.51 [95% CI: 0.26–1.03, p = 0.06]) and Latin America
(RR = 0.52 [95% CI: 0.27–1.01, p = 0.05]) demonstrated
reductions in NEC stage ≥2 incidence that was on the
threshold of significance. The only study location to show
significant reductions in all-cause mortality was Asia with a
RR of 0.35 (95% CI: 0.19–0.64, p = 0.0007). There were
insufficient numbers of studies to perform any meaningful
analysis for NEC-related mortality.
Subgroup analysis: Probiotics agent and incidence
of NEC stage ≥2
Subgroup analysis was performed to investigate differences
in efficacy in the reduction of NEC stage ≥2 incidence by
probiotic genus (Fig. 5A).
In trials where a combination of Bifidobacterium and
Lactobacillus species was used, there was a statistically
6 ©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd
Thomas et al.
significant reduction in the incidence of NEC stage ≥2.
Seven trials used a combination of Bifidobacterium and a
Lactobacillus species. The RCT by Fernandez-Carrocera
et al. (31), which used a combination of Bifidobacterium,
Lactobacillus and Streptococcus thermophilus was also
added to this group, as the concentration of Streptococcus
thermophilus was at least 100-fold lower than the other two
groups of bacteria in the probiotic mixture. The combined
RR across these eight trials (comprising of a total of 1623
neonates) was statistically significant at 0.41 (95% CI: 0.25–
0.66, p = 0.0003), and remained significant even after
removal of the trial by Ferandez-Carrocera et al.
However, pooled analysis of the five trials that utilised a
Lactobacillus species alone (RR = 0.63, 95% CI: 0.39–1.03,
p = 0.06) or the five trials that used Bifidobacterium species
in isolation (RR = 0.53, 95% CI: 0.22–1.26, p = 0.15),
revealed non-significant trends towards a reduced relative
risk of developing NEC stage ≥2. Pooled analysis of the two
trials (comprising a total of 624 neonates) using a combi-
nation of Bifidobacterium species and Streptococcus ther-
mophilus also resulted in a statistically non-significant
reduction in the incidence of NEC stage ≥2 with a RR of
0.29 (95% CI: 0.07–1.17, p = 0.08). There was no evidence
of significant benefit from the two trials that used Saccha-
romyces boulardii (RR=0.93, 95% CI: 0.45–1.94, p = 0.84).
Subgroup analysis: Probiotic agent and mortality
Subgroup analysis for the effect of probiotic genus on
mortality (Fig. 5B) and NEC-related mortality was per-
formed, although the latter was limited by a lack of power
due to the low number of included studies. Only the
subgroup using a combination of Bifidobacterium and
Lactobacillus species demonstrated a significant reduction
Thomas et al.
A B
NEC stage ≥ 2
Figure 4 Forest plot comparing incidence of (A) NEC stage ≥2 and (B) All-cause mortality in probiotic and control groups according to study location.
in all-cause mortality (RR = 0.47, 95% CI: 0.27–0.80,
p = 0.006). However, seven trials in this subgroup did not
report NEC-related mortality, so this could not be assessed.
Combined analysis of two trials that used a combination of
Bifidobacterium species and Streptococcus thermophilus
demonstrated a reduction in NEC-associated mortality
which was of borderline significance (RR = 0.32, 95% CI:
0.11–0.94, p = 0.04). No other significant associations
between probiotic genus and mortality were observed
(Table S4).
Meta-regression
Meta-regression analysis was used to evaluate the contri-
bution of potential treatment effect modifiers to the
between-study variability and overall effect size. Initial
univariate analysis showed non-significant trends for
increased effectiveness of probiotics in reducing NEC stage
≥2 with increases in birthweight, gestational age, exclusive
breastmilk feeding (defined as neonatal feeding exclusively
with breastmilk) and decreases in prenatal steroid exposure
(defined as any maternal antenatal exposure to corticos-
teroids irrespective of the dose, frequency and timing in
relation to delivery) and caesarean section in the popula-
tion. However, multivariate analysis revealed that the only
variables significantly associated with changes in the effec-
tiveness of the intervention (i.e. log risk ratio of NEC stage
©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd
Probiotics for preventing NEC in VLBW infants
All-cause deaths
≥2 incidence) were percentage rates of exclusive breastmilk
feeding (association coefficient 0.0134, p = 0.0168) and
prenatal steroid exposure (association coefficient 0.0158,
p = 0.0189). This implied that probiotics were more effec-
tive in cohorts where higher proportions of neonates were
exclusively breastfed and also in cohorts where lower
proportions of mothers received antenatal exposure to
steroids (Table 2).
Publication bias
The funnel plots for analysis on NEC incidence but not all-
cause mortality or NEC-related mortality displayed some
asymmetry (Fig. 6). This was quantified using the Egger’s
regression test, which was statistically significant for the
analysis relating to NEC incidence (p = 0.008) but not for
all-cause mortality (p = 0.06) or NEC-related mortality
(p = 0.71).
DISCUSSION
Our meta-analysis, which utilised data of over 7000 VLBW
infants from 23 RCTs, demonstrates that probiotics signif-
icantly reduce the incidence of NEC stage ≥2 and all-cause
mortality, with a trend towards reduction in NEC-related
mortality. Although the effect sizes are smaller than those
reported in earlier meta-analyses (Table 3), they are still
7
Probiotics for preventing NEC in VLBW infants Thomas et al.

A B
NEC stage ≥ 2 All-cause deaths

Figure 5 Forest plot comparing incidence of (A) NEC stage ≥ 2 and (B) All-cause mortality in probiotic and control groups according to probiotic agent.

Table 2 Multivariate regression model of log risk ratio of NEC stage ≥2 incidence on % Exclusive breastmilk feeding and % Prenatal steroid exposure
Covariate Coefficient Standard error 95% CI Z-value 2-sided p-value

Intercept 1.0498 0.6037 2.2330, 0.1334 1.74 0.0820

% Exclusive BreastMilk 0.0134 0.0056 0.0244, 0.0024 2.39 0.0168
% Prenatal steroid exposure 0.0158 0.0067 0.0026, 0.0291 2.35 0.0189

Test of model – Simultaneous test that all coefficients (excluding intercept) are zero: Q = 11.07, df = 2, p = 0.0040.
Null model – Total between-study variance (intercept only): Tau2 = 0.1363, I2 = 35.41%, Q = 20.13, df = 13, p = 0.0921.
Number of studies in analysis: 14.

substantial. We have also adopted a random-effects model
due to the heterogeneity observed. This is a more robust
method than the fixed-effects approaches used in previous
meta-analyses and yields wider confidence intervals, but
importantly the key findings remain highly statistically
significant.
Over the past decades, the search for a useful probiotic
agent for the prevention of NEC has included a myriad of
different probiotic combinations with varying degrees of
efficacy. Given the variation in results, we performed a
subgroup analysis with the aim of identifying differences in
efficacy based on probiotic agent. Only a few studies have
utilised the same probiotic formulation containing identical
strains and doses (Table S3). Identical preparations of
Dicloforâ (containing Lactobacillus rhamnosus) (41,44)
and BioGaia ABâ (containing Lactobacillus reuteri) (27,34)
were used in two trials each which demonstrated non-
significant reductions in NEC stage ≥2 incidence and
mortality. The same preparation of Infloranâ (containing
Lactobacillus acidophilus and Bifidobacterium bifidum)
was used in two trials (28,40), but showed contrasting
results. Very similar preparations of ABC Dophilusâ,
containing a combination of Bifidobacterium strains and
Streptococcus thermophilus, were utilised in two trials (29,
42), both of which resulted in statistically significant
reductions in the incidence of NEC stage ≥2. As only a
few studies utilised the same probiotic formulation with the
same strain(s), we were unable to perform a strain-specific
subgroup meta-analysis. Hence, we performed a genus-
specific subgroup analysis.
The subgroup analysis identified that only studies that
utilised a probiotic combination of Lactobacillus and
Bifidobacterium species showed statistically significant
reductions in both NEC incidence and mortality (Fig. 5).

8 ©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd
Thomas et al.
A NEC stage ≥ 2
Egger’s Test, p-value = 0.008
Figure 6 Funnel plots for (A) NEC stage ≥2, (B) All-cause mortality & (C) NEC-related mortality.
The evidence for this subgroup was particularly strong, as it
comprised of eight RCTs with data from more than 1600
neonates. Thus, it is likely that the overall efficacy signal
from probiotics in preventing NEC is being driven by
formulations containing this particular probiotic combina-
tion. It is not clear why this probiotic combination appears
to be the most effective. Current research indicates that a
dysbiosis with excessive amounts of gamma-proteobacteria
(mainly Enterobacteriaciae species) in relation to Firmi-
cutes (such as Lactobacilli), Actinobacteria (such as Bifi-
dobacteria) and Bacteroidetes precede the development of
NEC (46,47). Perhaps the introduction of the above
combination of probiotic agents is required to restore the
balance of the infant gut microbiome and prevent NEC
though interactions with the innate and adaptive immune
systems. Although the ideal combination of strains and the
optimal doses required to provide effect are not yet known,
it would be possible in the future to determine answers to
these once we have sufficient numbers of RCTs using
identical Lactobacillus and Bifidobacterium containing
formulations.
We also performed meta-regression analysis to identify
the contributions of other measured variables to the overall
effect size and between-study variability. Despite the low
©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd
Probiotics for preventing NEC in VLBW infants
B All-cause deaths
Egger’s Test, p-value = 0.06
C NEC-related deaths
Egger’s Test, p-value = 0.71
power due to the limited reporting of some studies, in
multivariate analysis we identified significant increases in
the effectiveness of probiotics in reducing NEC when used
in cohorts with increased rates of exclusive breastfeeding
and decreased levels of prenatal steroid exposure (Table 2).
These results indicate that breastmilk may act synergisti-
cally with probiotics to reduce the incidence of NEC. This
correlates with previous clinical trials which have shown
breastmilk to be protective against NEC (48), and basic
scientific research which has demonstrated that human
milk oligosaccharides found within breastmilk can facili-
tate growth of particular species of gut bacteria including
certain Bifidobacteria species (49). However, prenatal
steroids have been shown to also reduce the incidence of
NEC (50). One possible explanation why we found probi-
otics to be less effective in infants exposed to prenatal
steroids could be if both steroids and probiotics act through
the same biological pathways, thus diluting any additional
effect from probiotics. In fact previous research has shown
that both glucocorticoids and probiotic combinations of
Lactobacillus acidophilus and Bifidobacterium infantis
protect against NEC by attenuating excessive LPS and
IL-1 dependent IL-8 overexpression in the immature gut
(51,52).
9
Probiotics for preventing NEC in VLBW infants Thomas et al.

Table 3 Previous meta-analyses looking at probiotic supplementation in VLBW infants

Meta-analysis Number of studies Number of infants NEC stage ≥2 Death Sepsis NEC-related mortality

Deshpande (12) 7 1393 0.39 (0.20–0.65) 0.47 (0.30–0.73) 0.94 (0.74–1.20) Not calculated
Deshpande (11) 11 2176 0.35 (0.23–0.55) 0.42 (0.29, 0.62) 0.98 (0.81, 1.18) Not calculated
Wang (13) 20 3816 0.33 (0.24–0.46) 0.56 (0.43–0.73) 0.90 (0.71–1.15) Not calculated
AlFaleh (10) 17 4914 0.41 (0.31–0.56) 0.66 (0.53–0.82) 0.92 (0.81–1.04) 0.39 (0.18, 0.82)
Baucells (14) 9 3521 0.39 (0.26–0.57) 0.70 (0.52–0.93) 0.91 (0.78–1.06) 0.35 (0.14–0.89)
Lau & Chamberlain (9) 20 5982 0.51 (0.39–0.67) 0.73 (0.58–0.93) 0.92 (0.82–1.03) Not calculated
Aceti (45) 22 5912 0.48 (0.37–0.62) Not calculated Not calculated Not calculated

Table 4 Case reports of probiotic-associated bacteraemia/sepsis during the neonatal period

Post-natal Birth Day of Isolated
References age (weeks) weight (g) Probiotic Sepsis/Bacteraemia bacteria Management

Esaiassen et al. (53) <28 <1000 Infloran (LA + BLo) Day 8 BLo Antibiotic therapy, Laparotomy for NEC
Esaiassen et al. (53) <28 <1000 Infloran (LA + BLo) Day 12 BLo Antibiotic therapy
Esaiassen et al. (53) <28 <1000 Infloran (LA + BLo) Day 46 BLo Antibiotic therapy, Laparotomy for NEC
Bertelli et al. (54) 26 2/7 867 Infloran (LA + BLo) Day 14 BLo Antibiotic therapy, Laparotomy for small
bowel obstruction secondary to intussusception.
Bertelli et al. (54) 28 6/7 1090 Infloran (LA + BLo) Day 10 BLo Antibiotic therapy, Laparotomy for NEC
Zbinden et al. (55) 30 1200 Infloran (LA + BLo) Day 20 BLo No treatment required
Zbinden et al. (55) 28 850 Infloran (LA + BLo) Day 20 BLo No treatment required
Zbinden et al. (55) 29 1230 Infloran (LA + BLo) Day 11 BLo Antibiotic therapy, Laparotomy for NEC
Jenke et al. (56) 27 5/7 600 Infloran (LA + BLo) Day 18 BLo Antibiotic therapy
Guenther et al. (57) 28 935 E. coli NISSLE Day 10 E. coli NISSLE Antibiotic therapy

LA = Lactobacillus acidophilus; BLo = Bifidobacterium longum.

A meta-analysis to evaluate the impact of probiotic
supplementation on NEC in the ELBW infant population
was also performed, as this has been previously questioned
(53). Unlike the analysis on VLBW infants, our findings
showed no significant evidence of benefit with probiotic
supplementation in preventing NEC or death in ELBW
infants (Fig. 3). However, it is important to note that this
analysis was limited in that only five of the 23 RCTs
reported specific outcomes relating to ELBW infants.
Furthermore, meta-regression analysis, as previously
described, did not show a significant relationship between
birthweight and the effectiveness of probiotic supplemen-
tation. Hence, it would be premature to arrive at any
formal conclusions in this group at present based on these
findings alone.
A meta-analysis was also performed to assess for any
differences in effectiveness of probiotics according to the
geographical location of the study (Fig. 4), as environmen-
tal factors may potentially play a role in determining the
neonatal gut microbiome and in turn the efficacy of
probiotics (54). Only studies located in Asia and Australia
showed significant reductions in NEC stage ≥2. All-cause
mortality was only shown to be significantly reduced in
studies from Asia. Although interesting, these results are
hard to interpret as they may simply be a reflection of the
different probiotic agents utilised across trials rather than a
true effect of the location.
Aside from questions regarding efficacy, one of the
important barriers preventing the routine administration
of prophylactic probiotics in neonates is concerns regarding
its safety. None of the 3703 neonates given probiotics across
the 23 studies in this analysis developed short-term adverse
effects following exposure to probiotics. However, there
have been several case reports of non-fatal probiotic-
associated bacteraemia/sepsis in the neonatal period
(Table 4; 55–59). Most of these reported cases have
involved the culture and isolation of Bifidobacterium
longum strains originating from the probiotic agent Inflo-
ranâ. Additionally, little is known about the longer-term
consequences, if any, of probiotic supplementation. A
previous prospective three-year follow-up cohort study did
not find any differences in growth or neurodevelopment
outcomes in VLBW infants that had received probiotics
compared to placebo (60). Further studies will be required
to definitively identify whether there are any potential long-
term effects of probiotic supplementation.
Adding to the concerns regarding safety is that in many
countries, including the USA and UK, probiotics are not
licensed as pharmaceutical agents and thus not subjected to
stringent quality and safety regulatory mechanisms applied
to medicinal products. Instead, probiotics are considered as
food supplements in these countries and regulated only
against national food manufacturing standards. This is
particularly significant, given that in 2014 an infant

10 ©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd
Thomas et al.
developed fatal gastrointestinal mucormycosis following
probiotic therapy with ABC Dophilusâ (manufactured in
the USA) which had become contaminated with the fungus,
Rhizopus oryzae (61). However, it is worth noting that
certain probiotic formulations are manufactured in some
countries as medicinal products and thus subjected to
rigorous pharmaceutical regulations.
Despite our extensive search strategy, one limitation of
this study was the apparent publication bias in favour of the
intervention for the analysis on NEC incidence (Fig. 6).
This may have been due to a theoretical under reporting of
small studies in favour of placebo. We did not observe any
such bias for all-cause mortality or NEC-related mortality.
Another potential limitation could be the lack of analysis
for other outcomes such as late-onset sepsis and feeding.
However, we decided to streamline our search strategy and
solely focus our attention on NEC incidence and mortality.
CONCLUSION
Despite the controversy that characterises this topic, our
analysis shows with considerable statistical confidence that
probiotics prevent NEC and reduce mortality in VLBW
infants. We have identified that the most effective probiotic
formulations are those containing Lactobacillus and Bifi-
dobacterium species. Our findings also suggest that human
breastmilk feeding may act synergistically with probiotics to
protect against NEC, whilst prenatal steroid exposure may
attenuate the protective effects of probiotics. However,
there still remain important questions including the optimal
probiotic strains, doses and duration of therapy. Further-
more, there is a rare but definite risk of probiotic-associated
sepsis, and there may be potential long-term consequences
of probiotic supplementation that have not yet been
determined. Another important concern is that in many
countries probiotic agents are not manufactured under
stringent pharmaceutical regulations. Despite the consider-
able efficacy of probiotics, in our opinion, only once the
issues regarding strain selection and safety have been
addressed, and a standardised probiotic regimen has been
identified, would a shift from current clinical practice be
fully warranted. In spite of these challenges, we believe with
continued clinical and basic scientific research in this field,
it will be possible to one day realise the goal of identifying
an optimal probiotic strategy against NEC.
ACKNOWLEDGEMENTS
We thank Dr Vidheya Venkatesh and Dr Mithuna Urs for
ideas.
CONFLICT OF INTEREST
None reported.
FUNDING
No funding.
©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd
Probiotics for preventing NEC in VLBW infants
References
1. Neu J, Walker WA. Necrotizing enterocolitis. N Engl J Med
2011; 364: 255–64.
2. Obladen M. Necrotizing enterocolitis–150 years of fruitless
search for the cause. Neonatology 2009; 96: 203–10.
3. Holman RC, Stoll BJ, Curns AT, Yorita KL, Steiner CA,
Schonberger LB. Necrotising enterocolitis hospitalisations
among neonates in the United States. Paediatr Perinat
Epidemiol 2006; 20: 498–506.
4. Schmid O, Quaiser K. Uer eine besondere schwere verlaufende
Form von Enteritis beim saugling. Oesterr Z Kinderh 1953; 8:
114. Open Access Library. Available at: http://www.oalib.com/
references/14493243 (accessed on March 1, 2015).
5. Afrazi A, Sodhi CP, Richardson W, Neal M, Good M, Siggers R,
et al. New insights into the pathogenesis and treatment of
necrotizing enterocolitis: toll-like receptors and beyond.
Pediatr Res 2011; 69: 183–8.
6. Grishin A, Papillon S, Bell B, Wang J, Ford HR. The role of the
intestinal microbiota in the pathogenesis of necrotizing
enterocolitis. Semin Pediatr Surg 2013; 22: 69–75.
7. Lin H-Y, Chang JH, Chung M-Y, Lin H. Prevention of
necrotizing enterocolitis in preterm very low birth weight
infants: Is it feasible? J Formos Med Assoc 2014; 113: 490–7.
8. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B,
et al. Expert consensus document. The International Scientific
Association for Probiotics and Prebiotics consensus statement
on the scope and appropriate use of the term probiotic. Nat Rev
Gastroenterol Hepatol 2014; 11: 506–14.
9. Lau CSM, Chamberlain RS. Probiotic administration can
prevent necrotizing enterocolitis in preterm infants: a meta-
analysis. J Pediatr Surg 2015; 50: 1405–12.
10. AlFaleh K, Anabrees J. Probiotics for prevention of necrotizing
enterocolitis in preterm infants. Cochrane Database Syst Rev
2014; 4: CD005496.
11. Deshpande G, Rao S, Patole S, Bulsara M. Updated meta-
analysis of probiotics for preventing necrotizing enterocolitis in
preterm neonates. Pediatrics 2010; 125: 921–30.
12. Deshpande G, Rao S, Patole S. Probiotics for prevention
of necrotising enterocolitis in preterm neonates with very
low birthweight: a systematic review of randomised
controlled trials. Lancet (London, England) 2007; 369:
1614–20.
13. Wang Q, Dong J, Zhu Y. Probiotic supplement reduces risk of
necrotizing enterocolitis and mortality in preterm very low-
birth-weight infants: an updated meta-analysis of 20
randomized, controlled trials. J Pediatr Surg 2012; 47: 241–8.


14. Baucells BJ, Mercadal Hally M, Alvarez
nchez AT, Figueras
Sa
Aloy J. Probiotic associations in the prevention of necrotising
enterocolitis and the reduction of late-onset sepsis and
neonatal mortality in preterm infants under 1500 g: a
systematic review. An Pediatr (Barc) 2016; 85: 247–55.
15. Janvier A, Lantos J, Barrington K. The politics of probiotics:
probiotics, necrotizing enterocolitis and the ethics of neonatal
research. Acta Paediatr 2013; 102: 116–8.
16. Caplan M, Frost B. Myth: necrotizing enterocolitis: probiotics
will end the disease, and surgical intervention improves the
outcome. Semin Fetal Neonatal Med 2011; 16: 264–8.
17. Neu J, Shuster J. Nonadministration of routine probiotics
unethical—really? Pediatrics 2010; 126.
18. Bell MJ, Ternberg JL, Feigin RD, Keating JP, Marshall R,
Barton L, et al. Neonatal necrotizing enterocolitis. Therapeutic
decisions based upon clinical staging. Ann Surg 1978; 187: 1–7.
Available at: http://www.pubmedcentral.nih.gov/articlerende
r.fcgi?artid=1396409&tool=pmcentrez&rendertype=abstract
(accessed on February 28, 2015).
11
Probiotics for preventing NEC in VLBW infants
19. Higgins JPT GS (editors). Cochrane handbook for systematic
reviews of interventions version 5.1.0 [updated March 2011].
Cochrane Collab, 2011.
20. Borenstein M, Hedges LV, Higgins JPTRH. Introduction to
meta-analysis. 1st ed. Chichester: John Wiley Sons, 2009.
21. Kitajima H, Sumida Y, Tanaka R, Yuki N, Takayama H,
Fujimura M. Early administration of Bifidobacterium breve to
preterm infants: randomised controlled trial. Arch Dis Child
Fetal Neonatal Ed 1997; 76: F101–7.
22. Costeloe K, Hardy P, Juszczak E, Wilks M, Millar MR,
Berrington JE, et al. Bifidobacterium breve BBG-001 in very
preterm infants: a randomised controlled phase 3 trial. Lancet
2016; 387: 649–60.
€
23. Dilli D, Aydin B, Fettah ND, Ozyazıcı E, Beken S, Zenciroglu
A, et al. The ProPre-Save Study: effects of probiotics and
prebiotics alone or combined on necrotizing enterocolitis in
very low birth weight infants. J Pediatr 2015; 166(545–551): e1.
24. Hays S, Jacquot A, Gauthier H, Kempf C, Beissel A, Pidoux O,
et al. Probiotics and growth in preterm infants: a randomized
controlled trial. PREMAPRO study. Clin Nutr 2016; 35:
802–11.
25. Totsu S, Yamasaki C, Terahara M, Uchiyama A, Kusuda S.
Bifidobacterium and enteral feeding in preterm infants: cluster-
randomized trial. Pediatr Int 2014; 56: 714–9.
26. Patole S, Keil AD, Chang A, Nathan E, Doherty D, Simmer K,
et al. Effect of Bifidobacterium breve M-16V supplementation
on fecal bifidobacteria in preterm neonates - a randomised
double blind placebo controlled trial. PLoS ONE 2014; 9:
e89511.
27. Oncel MY, Sari FN, Arayici S, Guzoglu N, Erdeve O, Uras N,
et al. Lactobacillus Reuteri for the prevention of necrotising
enterocolitis in very low birthweight infants: a randomised
controlled trial. Arch Dis Child Fetal Neonatal Ed 2014; 99:
F110–5.
28. Saengtawesin V, Tangpolkaiwalsak R, Kanjanapattankul W.
Effect of oral probiotics supplementation in the prevention of
necrotizing enterocolitis among very low birth weight preterm
infants. J Med Assoc Thai 2014; 97(Suppl 6): S20–5.
29. Jacobs SE, Tobin JM, Opie GF, Donath S, Tabrizi SN, Pirotta
M, et al. Probiotic effects on late-onset sepsis in very preterm
infants: a randomized controlled trial. Pediatrics 2013; 132:
1055–62.
30. Demirel G, Erdeve O, Celik IH, Dilmen U. Saccharomyces
boulardii for prevention of necrotizing enterocolitis in preterm
infants: a randomized, controlled study. Acta Paediatr 2013;
102: 560–5.
31. Serce O, Benzer D, Gursoy T, Karatekin G, Ovali F. Efficacy of
Saccharomyces boulardii on necrotizing enterocolitis or sepsis
in very low birth weight infants: a randomised controlled trial.
Early Hum Dev 2013; 89: 1033–6.
32. Ferna
ndez-Carrocera LA, Solis-Herrera A, Cabanillas-Ayo
n M,
Gallardo-Sarmiento RC, Garc
ıa-P
erez CS, Montan ~ o-Rodr
ıguez
R, et al. Double-blind, randomised clinical assay to evaluate
the efficacy of probiotics in preterm newborns weighing less
than 1500 g in the prevention of necrotising enterocolitis. Arch
Dis Child Fetal Neonatal Ed 2013; 98: F5–9.
33. Al-Hosni M, Duenas M, Hawk M, Stewart LA, Borghese RA,
Cahoon M, et al. Probiotics-supplemented feeding in extremely
low-birth-weight infants. J Perinatol 2012; 32: 253–9.
34. Rojas MA, Lozano JM, Rojas MX, Rodriguez VA, Rondan MA,
Bastidas JA, et al. Prophylactic probiotics to prevent death and
nosocomial infection in preterm infants. Pediatrics 2012; 130:
1113–20.
35. Sari FN, Dizdar EA, Oguz S, Erdeve O, Uras N, Dilmen U.
Oral probiotics: Lactobacillus sporogenes for prevention of
necrotizing enterocolitis in very low-birth weight infants:
12
Thomas et al.
a randomized, controlled trial. Eur J Clin Nutr 2011; 65:
434–9.
36. Braga TD, Da Silva GAP, De Lira PIC, de Carvalho Lima M.
Efficacy of Bifidobacterium breve and Lactobacillus casei oral
supplementation on necrotizing enterocolitis in very-low-birth-
weight preterm infants: a double-blind, randomized, controlled
trial. Am J Clin Nutr 2011; 93: 81–6.
37. Mihatsch WA, Vossbeck S, Eikmanns B, Hoegel J, Pohlandt F.
Effect of Bifidobacterium lactis on the incidence of nosocomial
infections in very-low-birth-weight infants: a randomized
controlled trial. Neonatology 2010; 98: 156–63.
38. Roug
e C, Piloquet H, Butel M-J, Berger B, Rochat F, Ferraris L,
et al. Oral supplementation with probiotics in very-low-birth-
weight preterm infants: a randomized, double-blind, placebo-
controlled trial. Am J Clin Nutr 2009; 89: 1828–35.
39. Samanta M, Sarkar M, Ghosh P, Ghosh JK, Sinha MK,
Chatterjee S. Prophylactic probiotics for prevention of
necrotizing enterocolitis in very low birth weight newborns.
J Trop Pediatr 2009; 55: 128–31.
40. Lin H-C, Hsu C-H, Chen H-L, Chung M-Y, Hsu J-F, R-I Lien,
et al. Oral probiotics prevent necrotizing enterocolitis in very
low birth weight preterm infants: a multicenter, randomized,
controlled trial. Pediatrics 2008; 122: 693–700.
41. Manzoni P, Mostert M, Leonessa ML, Priolo C, Farina D,
Monetti C, et al. Oral supplementation with Lactobacillus
casei subspecies rhamnosus prevents enteric colonization by
Candida species in preterm neonates: a randomized study. Clin
Infect Dis 2006; 42: 1735–42.
42. Bin-Nun A, Bromiker R, Wilschanski M, Kaplan M, Rudensky
B, Caplan M, et al. Oral probiotics prevent necrotizing
enterocolitis in very low birth weight neonates. J Pediatr 2005;
147: 192–6.
43. Lin H-C, Su B-H, Chen A-C, Lin T-W, Tsai C-H, Yeh T-F, et al.
Oral probiotics reduce the incidence and severity of
necrotizing enterocolitis in very low birth weight infants.
Pediatrics 2005; 115: 1–4.
44. Dani C, Biadaioli R, Bertini G, Martelli E, Rubaltelli F. Probiotics
feeding in prevention of urinary tract infection, bacterial sepsis
and necrotizing enterocolitis in preterm infants. A prospective
double-blind study. Biol Neonate 2002; 82: 103–8.
45. Aceti A, Gori D, Barone G, Callegari ML, Di Mauro A, Fantini
MP, et al. Probiotics for prevention of necrotizing enterocolitis
in preterm infants: systematic review and meta-analysis. Ital J
Pediatr 2015; 41: 89.
46. Torrazza RM, Neu J. The altered gut microbiome and
necrotizing enterocolitis. Clin Perinatol 2013; 40: 93–108.
47. Elgin TG, Kern SL, McElroy SJ. Development of the neonatal
intestinal microbiome and its association with necrotizing
enterocolitis. Clin Ther 2016; 38: 706–15.
48. Quigley M, McGuire W. Formula versus donor breast milk for
feeding preterm or low birth weight infants. In: M Quigley,
editor. Cochrane database of systematic reviews. John Wiley &
Sons Ltd: Chichester, UK, 2014. https://doi.org/10.1002/
14651858.cd002971.pub3.
49. Ward RE, Nin ~ onuevo M, Mills DA, Lebrilla CB, German JB. In
vitro fermentation of breast milk oligosaccharides by
Bifidobacterium infantis and Lactobacillus gasseri. Appl
Environ Microbiol 2006; 72: 4497–9.
50. Wong D, Abdel-Latif M, Kent A. Antenatal steroid exposure
and outcomes of very premature infants: a regional cohort
study. Arch Dis Child Fetal Neonatal Ed 2014; 99: F12–20.
51. Nanthakumar NN, Young C, Ko JS, Meng D, Chen J, Buie T,
et al. Glucocorticoid responsiveness in developing human
intestine: possible role in prevention of necrotizing
enterocolitis. Am J Physiol Gastrointest Liver Physiol 2005;
288: G85–92.
©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd
Thomas et al.
52. Ganguli K, Meng D, Rautava S, Lu L, Walker WA,
Nanthakumar N. Probiotics prevent necrotizing enterocolitis
by modulating enterocyte genes that regulate innate immune-
mediated inflammation. AJP Gastrointest Liver Physiol 2013;
304: G132–41.
53. Abrahamsson TR, Rautava S, Moore AM, Neu J, Sherman PM.
The time for a confirmative necrotizing enterocolitis probiotics
prevention trial in the extremely low birth weight infant in
North America is now! The Journal of Pediatrics 2014; 165:
389–94.
54. Munyaka PM, Khafipour E, Ghia J-E. External influence of
early childhood establishment of gut microbiota and
subsequent health implications. Front Pediatr 2014; 2: 109.
55. Esaiassen E, Cavanagh P, Hjerde E, Simonsen GS, Støen R,
Klingenberg C. Bifidobacterium longum subspecies infantis
bacteremia in 3 extremely preterm infants receiving probiotics.
Emerg Infect Dis 2016; 22(9): 1664–6.
56. Bertelli C, Pillonel T, Torregrossa A, Prodhom G, Fischer CJ,
Greub G, et al. Bifidobacterium longum bacteremia in preterm
infants receiving probiotics. Clin Infect Dis 2015; 60: 924–7.
57. Zbinden A, Zbinden R, Berger C, Arlettaz R. Case series of
Bifidobacterium longum bacteremia in three preterm infants
on probiotic therapy. Neonatology. 2015; 107: 56–9.
58. Jenke A, Ruf E-M, Hoppe T, Heldmann M, Wirth S.
Bifidobacterium septicaemia in an extremely low-birthweight
infant under probiotic therapy. Arch Dis Child Fetal Neonatal
Ed 2012; 97: F217–8.
59. Guenther K, Straube E, Pfister W, Guenther A, Huebler A.
Sever sepsis after probiotic treatment with Escherichia coli
NISSLE 1917. Pediatr Infect Dis J. 2010; 29: 188–9.
60. Chou I-C, Kuo H-T, Chang J-S, Wu S-F, Chiu H-Y, Su B-H,
et al. Lack of effects of oral probiotics on growth and
neurodevelopmental outcomes in preterm very low birth
weight infants. J Pediatr 2010; 156: 393–6.
61. Vallabhaneni S, Walker TA, Lockhart SR, Ng D, Chiller T,
Melchreit R, et al. Notes from the field: fatal gastrointestinal
mucormycosis in a premature infant associated with a
contaminated dietary supplement–Connecticut, 2014. MMWR
Morb Mortal Wkly Rep 2015; 64: 155–6.
62. Tewari VV, Dubey SK, Gupta G. Bacillus clausii for
prevention of late-onset sepsis in preterm infants: a
randomized controlled trial. J Trop Pediatr 2015; 61: 377–85.
63. Dutta S, Ray P, Narang A. Comparison of stool colonization in
premature infants by three dose regimes of a probiotic
combination: a randomized controlled trial. Am J Perinatol
2015; 32: 733–40.
64. Savino F, De Marco A, Ceratto S, Mostert M. Fecal
calprotectin during treatment of severe infantile colic with
Lactobacillus reuteri DSM 17938: a randomized, double-blind,
placebo-controlled trial. Pediatrics 2015; 135(Supplement 1):
S5–6.
65. Sharma B, Srivastava S, Singh N, Sachdev V, Kapur S, Saraya
A. Role of probiotics on gut permeability and endotoxemia in
patients with acute pancreatitis. J Clin Gastroenterol 2011; 45:
442–8.
66. Hoyos AB. Reduced incidence of necrotizing enterocolitis
associated with enteral administration of Lactobacillus
©2017 Foundation Acta Pædiatrica. Published by John Wiley & Sons Ltd
Probiotics for preventing NEC in VLBW infants
acidophilus and Bifidobacterium infantis to neonates in an
intensive care unit. Int J Infect Dis 1999; 3: 197–202.
67. Costalos C, Skouteri V, Gounaris A, Sevastiadou S,
Triandafilidou A, Ekonomidou C, et al. Enteral feeding of
premature infants with Saccharomyces boulardii. Early Hum
Dev 2003; 74: 89–96.
68. Stratiki Z, Costalos C, Sevastiadou S, Kastanidou O,
Skouroliakou M, Petrohilou V, et al. The effect of a
bifidobacter supplemented bovine milk on intestinal
permeability of preterm infants. Early Hum Dev 2007; 83:
575–9.
69. Awad H, Mokhtar H, Imam SS, Gad GI, Hafez H, Aboushady N.
Comparison between killed and living probiotic usage versus
placebo for the prevention of necrotizing enterocolitis and sepsis
in neonates. Pakistan J Biol Sci PJBS 2010; 13: 253–62.
70. Hua X-T, Tang J, Mu D-Z. Effect of oral administration of
probiotics on intestinal colonization with drug-resistant
bacteria in preterm infants. Zhongguo Dang Dai Er Ke Za Zhi
2014; 16: 606–9.
71. Manzoni P, Rinaldi M, Cattani S, Pugni L, Romeo MG,
Messner H, et al. Bovine lactoferrin supplementation for
prevention of late-onset sepsis in very low-birth-weight
neonates: a randomized trial. JAMA 2009; 302: 1421–8.
72. Manzoni P, Stolfi I, Messner H, Cattani S, Laforgia N, Romeo
MG, et al. Bovine lactoferrin prevents invasive fungal
infections in very low birth weight infants: a randomized
controlled trial. Pediatrics 2012; 129: 116–23.
73. Manzoni P, Meyer M, Stolfi I, Rinaldi M, Cattani S, Pugni L,
et al. Bovine lactoferrin supplementation for prevention of
necrotizing enterocolitis in very-low-birth-weight neonates: a
randomized clinical trial. Early Hum Dev 2014; 90(Suppl 1):
S60–5.
74. Underwood MA, Kalanetra KM, Bokulich NA, Lewis ZT,
Mirmiran M, Tancredi DJ, et al. A comparison of two probiotic
strains of bifidobacteria in premature infants. J Pediatr 2013;
163(1585–1591): e9.
75. Van Niekerk E, Nel DG, Blaauw R, Kirsten GF. Probiotics
reduce necrotizing enterocolitis severity in HIV-exposed
premature infants. J Trop Pediatr 2015; 61: 155–64.
76. Benor S, Marom R, Ben Tov A, Tov AB, Armoni Domany K,
Domany KA, et al. Probiotic supplementation in mothers of
very low birth weight infants. Am J Perinatol 2014; 31: 497–
504.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the
online version of this article:
Table S1 Jadad Scores.
Table S2 Neonatal and maternal characteristics of probiotic
group.
Table S3 Study probiotic protocols.
Table S4 Results of sub-meta-analysis according to probi-
otic genus.
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