See corresponding editorial on page 1411.

Benefits of probiotics on enteral nutrition in preterm neonates:

a systematic review13
Gayatri Athalye-Jape, Girish Deshpande, Shripada Rao, and Sanjay Patole

Keywords enteral feeds, review, infants, neonates, nutrition, preterm, probiotic supplementation

INTRODUCTION

A recent updated Cochrane review (1) of randomized controlled trials (RCTs)4 (24 RCTs; 5529 neonates) confirmed
previous findings that probiotic supplementation significantly
reduces risk of stage II or greater necrotizing enterocolitis
(NEC) (RR: 0.43, 95% CI: 0.33,0.56) and all-cause mortality
(RR: 0.65; 95% CI: 0.52, 0.81) in preterm neonates. These results reemphasized previous recommendations that a change in
practice in favor of probiotic supplementation is needed (14).
However, the debate about risks compared with benefits of
probiotic supplementation in preterm neonates continues (57).
With consideration of the evidence from RCTs (1) and reports

1508

on the routine use of probiotics (811), we believe that it is time to

shift the focus from the well-reported and -accepted benefits of
probiotics in reducing risk of NEC and mortality to improving other
enteral nutritionrelated outcomes (e.g., facilitating feed tolerance).
A nutritional deficit and poor growth in the early postnatal
period are associated with long-term neurodevelopmental impairment, short stature, and metabolic disorders in preterm neonates
(1217). Extrauterine growth restriction is common at discharge in
extremely low-birth-weight infants (birth weight ,1000 g) and
relates to their significant cumulative caloric and protein deficits
during hospitalization and slower growth velocity associated with
complications of prematurity (1823). Therefore, the optimization
of nutrition in the early postnatal life of a preterm neonate is
a priority. Probiotics are known to have various potentially beneficial effects on gut function (24) and maturity (25). Therefore, we
aimed to systematically review RCTs that reporting effect of
probiotic supplementation on enteral nutrition in preterm neonates.
To our knowledge, previous systematic reviews have not adequately addressed this important issue (1, 2628).
Our primary outcome of interest was the time to establish full
enteral feeds (TFEFs). Secondary outcomes included the time to
regain birth weight, number of episodes of feed intolerance, and
duration of hospitalization.
METHODS

Guidelines from the Cochrane Neonatal Review Group, Centre

for Reviews and Dissemination (29), and the Preferred Reporting
Items for Systematic Reviews and Meta-Analyses statement were
1

From the Department of Neonatal Paediatrics, King Edward Memorial

Hospital, Perth, Western Australia (GA-J, SR, and SP); the Department of
Neonatal Paediatrics, Princess Margaret Hospital for Children, Perth, Western
Australia (GA-J, SR, and SP); the Centre for Neonatal Research and Education, School of Paediatrics and Child Health, University of Western Australia,
Perth, Western Australia (GA-J, SR, and SP); the Department of Neonatal
Paediatrics, Nepean Hospital, Sydney, Australia (GD); and Nepean Clinical
School, University of Sydney, Sydney, Australia (GD).
2
No funding or sponsorship was received for this study.
3
Address correspondence to S Patole, Department of Neonatal Paediatrics,
King Edward Memorial Hospital for Women, 374 Bagot Road, Subiaco,
Perth 6008, Western Australia. E-mail: sanjay.patole@health.wa.gov.au.
4
Abbreviations used: DSM, Deutsche Sammlung von Mikroorganismen;
NEC, necrotizing enterocolitis; RCT, randomized controlled trial; ROB, risk
of bias; SCFA, short-chain fatty acid; TFEF, time to full enteral feed.
Received June 13, 2014. Accepted for publication August 29, 2014.
First published online November 5, 2014; doi: 10.3945/ajcn.114.092551.

Am J Clin Nutr 2014;100:150819. Printed in USA. 2014 American Society for Nutrition

Downloaded from ajcn.nutrition.org by guest on October 4, 2016

ABSTRACT
Introduction: The optimization of enteral nutrition is a priority in
preterm neonates worldwide. Probiotics are known to improve gut
maturity and function in preterm neonates. To our knowledge, previous systematic reviews have not adequately assessed the effects of
probiotic supplementation on enteral nutrition in preterm neonates.
Objective: We assessed the evidence on effects of probiotics on
enteral nutrition in preterm neonates.
Design: A systematic review of randomized controlled trials (RCTs) of
probiotic supplementation in preterm (gestation ,37 wk) or low-birthweight (birth weight ,2500 g) neonates was conducted. With the use of
the Cochrane Neonatal Review Group strategy, we searched the
Cochrane Central Register of Controlled Trials, PubMed, EMBASE, and
Cumulative Index of Nursing and Allied Health Literature databases and
proceedings of Pediatric Academic Society meetings in April 2014.
Results: A total of 25 RCTs (n = 5895) were included in the review.
A meta-analysis (random-effects model) of data from 19 of 25 trials
(n = 4527) estimated that the time to full enteral feeds was shorter in
the probiotic group (mean difference: 21.54 d; 95% CI: 22.75,
20.32 d; P , 0.00001, I2 = 93%). Other benefits included fewer
episodes of feed intolerance, better weight gain and growth velocity,
decreased transition time from orogastric to breast feeds, and increased postprandial mesenteric flow. There were no adverse effects
of probiotic supplementation.
Conclusions: Probiotics reduced the time to full enteral feeds in
preterm neonates. Additional research is necessary to assess the
optimal dose, duration, and probiotic strain or strains used specifically for facilitating enteral nutrition in this population.
Am J
Clin Nutr 2014;100:150819.

PROBIOTICS AND NUTRITION IN PRETERM NEONATES

1509

followed for undertaking and reporting this systematic review

and meta-analysis (30).

data by using a standardized data-collection form. Inconsistencies

were sorted out by discussion with all authors.

Types of studies

Assessment of risk of bias

Only RCTs were included. Retrospective studies, prospective

observational studies, narrative reviews, letters, editorials, and
commentaries were excluded but read to identify potential studies.

Risk of bias (ROB) was assessed by using the Cochrane Risk

of Bias Assessment Tool (31). Two authors (GA-J and SR) independently assessed ROB in all domains including randomnumber generation, allocation concealment, blinding of intervention
and outcome assessors, completeness of follow-up, selectivity of
reporting, and other potential sources of bias. For each domain, the
ROB was assessed as low risk, high risk, or unclear risk based on the
guidelines of the Cochrane Collaboration (31) as follows:

Types of participants
Preterm neonates born at a gestational age ,37 wk or of low
birth weight (,2500 g) were included.
Intervention and comparison

Outcomes
Studies were included if they reported at least one of the
following outcomes:
1) TFEFs.
2) Other nutritional outcomes such as the time to regain birth
weight, anthropometric measure, extrauterine growth restriction, and feed intolerance (vomiting, abdominal distension, and gastric aspirates).
Review methods
Search strategy
The Cochrane Central Register of Controlled Trials (through
April 2014; www.thecochranelibrary.com), PubMed (19662014;
www.ncbi.nlm.nih.gov), EMBASE via Ovid (19802014; http://
ovidsp.tx.ovid.com), and Cumulative Index of Nursing and Allied
Health Literature (19802014; http://www.ebscohost.com/academic/
the-cinahl-database) databases and E-Abstracts from the Pediatric
Academic Society meetings (20002014; www.abstracts2view.com/
pasall) were searched in April 2014. Reference lists of identified
studies and key review articles were also searched. No language
restriction was applied.
PubMed was searched by using the strategy for our previous
systematic review (26) with the following Medical Subject
Headings terms: [Infant, Premature], OR [Infant, Very Low Birth
Weight] OR [Infant, Low Birth Weight] OR [Infant, Extremely
Low Birth Weight] OR Infant, Small for gestational age] AND
[Randomized Controlled Trials] OR [Controlled Clinical Trials]
OR [Clinical Trials] AND [Probiotic]. The term Probiotic was
replaced by Bifidobacterium OR Lactobacillus OR Saccharomyces for additional refined search.
Data extraction
All authors searched the literature independently and assessed
inclusion criteria. Authors GA-J and GD independently extracted

1) Random sequence generation: The ROB in this domain was

assessed as low if random number tables or computergenerated random numbers were used. The risk was assessed as high if any other methods were used for random
sequence generation. The risk was assessed as unclear
when this information was not given in a clear way.
2) Allocation concealment: The ROB in this domain was assessed as low if central randomization by a third party was
used. Serially numbered, opaque, sealed, and sequentially
opened envelopes were also considered as low risk. The risk
was assessed as high if unsealed or transparent envelopes
were used or when they were not opened sequentially. The
risk was assessed as unclear when this information was not
given in a clear way.
3) Blinding of participants and personnel: The ROB in this domain was assessed as low if the authors clearly described that
participants and personnel (e.g., health care providers, investigators, parents) were blinded. The risk was assessed as high
if some or all of them were not blinded. The risk was assessed
as unclear when this information was not given in a clear way.
4) Blinding of outcome assessment: The ROB in this domain
was assessed as low if the authors clearly described that

FIGURE 1 Flow diagram of study-selection process. PubMed, www.ncbi.

nlm.nih.gov; Embase (via Ovid), http://ovidsp.tx.ovid.com; Cumulative Index
of Nursing and Allied Health Literature (CINAHL), http://www.ebscohost.
com/academic/the-cinahl-database; Cochrane Central Register of Controlled Trials (Cochrane), www.thecochranelibrary.com. RCT, randomized controlled trial.

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Studies that compared enteral administration of any probiotic

or probiotics commenced within the first 10 d of life and continued $7 d compared with a placebo or controls were eligible
for inclusion. For RCTs in which data on nutritional outcomes
were not available, authors were contacted. If there was no response, such studies were excluded. Studies that compared one
type of probiotic with another and those that compared different
doses of probiotics were not included.

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ATHALYE-JAPE ET AL.

TABLE 1
Nutritional outcomes in clinical trials of probiotic supplementation in preterm neonates1
(No. of study) first author,
year of publication
(reference)
1) Kitajima, 1997 (37)

2) Costalos, 2003 (38)

3) Bin-Nun, 2005 (39)

5) Stratiki, 2007 (41)

6) Lin, 2008 (42)

7) Samanta, 2008 (43)

8) Rouge, 2009 (44)

9) Braga, 2010 (45)

n = 91 (study: n = 45 compared with placebo: n = 46). Intervention: BBG compared with placebo (distilled water)
Type of milk: EBM/formula; type of delivery: data not available
Primary outcome: gut colonization with BBG
Nutritional outcomes: less air aspirated in first 4 wk of life and less vomiting and apneas in the BBG group (P . 0.05); colonized
infants established feeding earlier and had greater weight gain in the fourth and eighth weeks of life
n = 87 (study: n = 51 compared with placebo: n = 36). Intervention: Saccharomyces boulardii compared with maltodextrin
Type of milk: EBM. Type of delivery: CS, 49% (study group) compared with 38% (placebo group)
Primary outcome: tolerance to S. boulardiisupplemented formula, fecal flora analysis, intestinal D xylose absorption, and fecal
lipid excretion (80% power)
Secondary outcomes: incidence of NEC, 9.8% compared with 16% (P = 0.5); incidence of sepsis, 5.8% compared with 8.3%
(P = 0.7)
Nutritional outcome: median TFEF, 9.3 compared with 9.9 d (P . 0.1); median (IQR) weight gain (g/wk), 163.5 (17.7) compared
with 155.8 (16.5) (P . 0.05)
n = 145 (study: 72 n = compared with controls: n = 73). Intervention: (Bifidobacterium infantis + Streptococcus thermophilus +
Bifidobacterium bifidus) compared with no probiotic
Type of milk: EBM/formula. Type of delivery: 78% (LSCS in study group) compared with 78% (LSCS in controls)
Primary outcome: at least stage II NEC (80% power)
Nutritional outcomes: TFEF similar in both groups (P = 0.13). Mean (6SD) cumulative weight gain by 6 wk: 691 6 208 g in
probiotic group compared with 594 6 239 g in control group (P . 0.05)
n = 80 (study: 39 compared with controls: 40). Intervention: LGG compared with no probiotic
Type of milk: EBM/PDHM. Type of delivery: VD, 30% (study group) compared with 35% (controls)
Primary outcome: incidence of enteric fungal colonization (80% power)
Nutritional outcomes: mean (6SD) TFEF: 15 6 8 compared with 17 6 9 d (P = 0.15); duration of hospital stay: 30 6 28
compared with 35 6 30 d (P = 0.2)
n = 75 (study: 41 compared with controls: 34). Intervention: Bifidobacter lactissupplemented preterm formula compared with
preterm formula
Type of milk: preterm formula. Type of delivery: CS, 36.5% (study group) compared with 35% (controls)
Primary outcome: intestinal permeability
Nutritional outcomes [median (range)]: weight gain (g/d), 28.3 (1238) compared with 30 (1040) (P = 0.144); length gain (cm/wk).
1.4 (03) compared with 1.5 (03.5) (P = 0.271); head growth (cm/wk), 1.1 (0.451.9) compared with 0.9 (02) (P = 0.001); TFEFs
(d), 10 (052) compared with 10 (030) (P = 0.615); time to regain birth weight (d): 10 (420) compared with 13 (624)
(P = 0.072)
n = 434 (study: 217 compared with controls: 217). Intervention: (Lactobacillus acidophilus + Bifidobacterium bifidum) compared
with no probiotic
Type of milk: EBM/formula. Type of delivery: CS, 69.6% (study group) compared with 63.3% (controls)
Primary outcomes: death or NEC of at least stage II (P = 0.002) (power: 90%)
Nutritional outcomes (means 6 SDs): TFEFs, 29.8 6 19.7 compared with 27.0 6 18.7 d (P = 0.13); weight gain on day14 (53.4 6
53.1 compared with 52.7 6 59.8 g), day 28 (103.5 6 70 compared with 103.4 6 78.8 g), and day 42 (131.9 6 89.7 compared
with 139.7 6 85.6 g); hospital stay, 46.4 6 24.2 compared with 43.3 6 21.0 d (P = 0.16)
n = 186 (study: n = 91 compared with controls: n = 95). Intervention: probiotic mixture (B. infantis + B. bifidum + Bifidobacterium
longum + L. acidophilus) compared with no probiotic
Type of milk: EBM. Type of delivery: CS, 46.15% (study group) compared with 49.47% (controls)
Primary outcomes: feed tolerance (TFEFs), length of stay, comorbidities such as NEC, sepsis, and death due to NEC/sepsis
Nutritional outcomes: mean (6SD) TFEF lower in study group (13.76 6 2.28 d) than in controls (19.2 6 2.02 d) (P , 0.001),
mean (6SD) duration of hospital stay lower in study group (17.17 6 3.23 d) than controls (24.07 6 4 d) (P , 0.001), incidence
of NEC lower in study group (1.1%) than controls (15.8%) (P = 0.042) but no significant difference for NEC of at least stage II
(P = 0.62), lower culture-proven sepsis in study group (14.3%) than controls (29.5%) (P = 0.02), and lower death rate in study
group (4.4%) than controls (14.7%) (P = 0.032)
n = 94 (study: 45 compared with placebo: 49). Intervention: (B. longum + Lactobacillus rhamnosus GG) compared with placebo
(maltodextrin)
Type of milk: EBM/formula. Type of delivery: CS, 62.2% (study group) compared with 71.4% (placebo group)
Primary outcome: Percentage of age of infants receiving .50% of feeds via enteral route at day 14 of life (57.8% compared with
57.1%; P = 0.95) (80% power)
Nutritional outcomes: median TFEF, 16 compared with 26 d (P = 0.04)
n = 231 (study: n = 119 compared with controls: n = 112). Intervention: Yakult LB (Lactobacillus casei + Bifidobacterium breve)
compared with no probiotic
Type of milk: EBM/PDHM. Type of delivery, CS 53.8% (study group) compared with 49.1% (controls)
Primary outcome: confirmed NEC of at least stage II (80% power)
Nutritional outcomes: mean (6SD) TFEF, 15.2 6 5.2 compared with 17.4 6 5.7 d (P = 0.02); weight recovery in 14 d not much
different in study and control group (P = 0.82). Transition time from orogastric to breastfeeds: lesser in probiotic group
(P = 0.03)
(Continued)

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4) Manzoni, 2006 (40)

Study characteristics

PROBIOTICS AND NUTRITION IN PRETERM NEONATES

1511

TABLE 1 (Continued )
(No. of study) first author,
year of publication
(reference)

Study characteristics

10) Hikaru, 2010 (46)

(Continued)

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n = 208 (study: n = 108 compared with controls: n = 100). Intervention: B. breve compared with no probiotic
Type of milk: EBM/formula. Type of delivery: data not available
Primary outcome: incidence rate of infection and sepsis
Nutritional outcomes (mean 6 SD): TFEF, 13.9 6 6.89 compared with 19.6 6 16.1 d (P , 0.01); weight on original EDD:
2412 6 441.5 (intervention) compared with 2113 6 58.9 g (controls) (P = 0.01); duration of hospital stay: 91.8 6 54.1
compared with 95.7 6 47.4 d (P = NS)
11) Mihatsch, 2010 (47) n = 183 (study: n = 93 compared with placebo: n = 90). Intervention: B. lactis compared with placebo
Type of milk: EBM/formula. Type of delivery: VD, 30% (study group) compared with 31% (placebo group)
Primary outcome: incidence density of nosocomial infections from days 742 of life after the initiation of enteral feeding (power:
80%); no significant effect on incidence of NEC of at least stage II (2% compared with 4%; P = NS)
Nutritional outcomes: no difference in mean (6SD) TFEFs (17.9 6 6.8 compared with 18.0 6 7.4 d; P = NS)
12) Al Hosni, 2011 (48) n = 101 (study: n = 50 compared with controls: n = 51). Intervention: (L. rhamnosus GG + B. infantis) compared with no
probiotics
Type of milk: EBM. Type of delivery: CS, 44% (study group) compared with 59% (controls)
Primary outcome: percentage of infants below 10th percentile at 34 wk PMA (power: 80%)
Nutritional outcome: better mean (6SD) growth velocity in study group (14.9 6 6.5 compared with 12.6 6 4.5 g/d in controls; P =
0.05). Similar daily weight gain in both groups (P = 0.06) and no significant difference in weights ,10th percentile at 34 wk
PMA (27% compared with 28%; P = 0.83)
13) Chrzanowskan = 47 (study: n = 21 compared with placebo: n = 26). Intervention: LGG compared with maltodextrin
Liszewska, 2011 (49) Type of milk: formula. Type of delivery: VD, 23% (study group) compared with 34% (controls)
Primary and nutritional outcome: presence of LGG colonization in stools, somatic growth, and length of hospital stay;
(80% power)
Nutritional outcome: weight gain at discharge (P = 0.567); mean duration of hospital stay, 49.9 compared with 46 d (P = 0.421)
14) Romeo, 2011 (50)
n = 249 (study: Lactobacillus reuteri 83 + L. rhamnosus, n = 83 compared with controls: n = 83). Intervention: (L. reuteri + L.
rhamnosus) compared with no probiotics
Type of milk: EBM/formula. Type of delivery: CS, 94% (L. reuteri group) compared with 86% (L. rhamnosus group) compared
with 93% (controls)
Primary outcomes: incidence of enteric colonization by Candida, LOS, neurological outcome at 12-mo corrected gestational age
(Hammersmith Infant Neurological Examination at corrected 12 mo of age)
Nutritional outcomes: lesser gastrointestinal symptoms (reflux, vomiting, abdominal distension) in L. reuteri group compared with
L. rhamnosus and controls (P , 0.05 for .1500 g and total population), lesser mean (6SD) hospital stay in L. reuteri group
compared with L. rhamnosus and controls (P , 0.05; 17.8 6 7.9 d for L. reuteri, 26.9 6 15.7 d for L. rhamnosus, and 31.3 6
16.3 d for controls)
15) Sari, 2011 (51)
n = 221 (study: n = 110 compared with controls: n = 111). Intervention: Lactobacillus sporogenes compared with no probiotic
Type of milk: EBM/formula. Type of delivery: CS, 67.3% (study group) compared with 75.7% (controls)
Primary outcome: death or NEC of at least stage II (P = 0.515) (power: 80%)
Secondary outcomes: culture-proven sepsis without NEC, grade 34 IVH (P = 0.983)
Nutritional outcomes: feed intolerance, 44.5% compared with 63.1% (P = 0.006); feeding amount at 14 d (P = 0.539), 28 d
(P = 0.099), and 42 d (P = 0.468); mean (6SD) TFEFs, 17.3 6 8.7 compared with 18.3 6 9.8 d (P = 0.438); mean (6SD)
weight gain at 14 d (3.7 6 7.1 compared with 3.7 6 6 g; P = 0.977), 28 d (10.0 6 5.1 compared with 10.5 6 5.2 g; P = 0.555),
and 42 d (12.6 6 4.3 compared with 12.3 6 5 g; P = 0.769); median duration of hospital stay, 34.5 compared with 30 d (P = 0.919)
16) Rojas, 2012 (52)
n = 750 (study: 372 compared with placebo: 378). Intervention: L. reuteri compared with placebo
Type of milk: EBM/formula. Type of delivery: noninstrumental VD, 16% (study) compared with 17% (placebo); instrumental VD,
0% (study) compared with 0.5% (placebo); elective CS, 18% (study) compared with 17% (placebo); nonselective CS,
65% (study) compared with 65% (placebo)
Primary outcome: death or nosocomial infections (power: 90%)
Nutritional outcomes: lesser episodes of feed intolerance: 7% compared with 10.6%; P = 0.08 with significant effects observed
in subjects #1500 g (17 episodes in study group compared with 31 episodes in control group; P = 0.04); similar durations
of hospitalization in both groups (20 d) (P = 0.53); for TFEFs, P = 0.134
17A)2 Demirel, 2013 (53) n = 179 (study: n = 81 compared with controls: n = 98). Intervention: S. boulardii compared with no probiotic
Type of milk: EBM/formula. Type of delivery: CS, 79.1% (study group) compared with 82.2% (controls)
Primary outcome (mean 6 SD): effect of probiotic on the course of indirect hyperbilirubinemia and duration of phototherapy (1.9
6 0.86 compared with 2.6 6 0.91 d; P , 0.05)
Nutritional outcome: lesser feeding intolerance in study group (20.9% compared with 47.9%; P = 0.00)
17B) Demirel, 2013 (54) n = 181 (probiotic: n = 91 compared with controls: n = 90). Intervention: S. boulardii compared with nystatin (control)
Type of milk: EBM/formula. Type of delivery: CS, 79.1% (study group) compared with 82.2% (controls)
Primary outcome: incidence of fungal colonization of skin and invasive fungal infection (power: 80%)
Secondary outcomes: incidence of sepsis, mortality, necrotizing enterocolitis, retinopathy of prematurity, severe IVH, and
bronchopulmonary dysplasia
Nutritional outcome: incidence of feeding intolerance in study group (at least one episode): 20.5% compared with 44.9% (P = 0.001)

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ATHALYE-JAPE ET AL.

TABLE 1 (Continued )
(No. of study) first author,
year of publication
(reference)
17C) Demirel, 2013 (55)

18) Fernandez-Carrocera,
2013 (56)

19) Havranek, 2013 (57)

21) Serce, 2013 (59)

22) Oncel, 2014 (60)

23) Patole, 2014 (61)

24) Roy, 2014 (62)

n = 271 (study: n = 135 compared with controls: n = 136). Intervention: S. boulardii compared with no probiotic
Type of milk: EBM/formula. Type of delivery: CS, 79.1% (study group) compared with 82.2% (controls)
Primary outcome: NEC of at least stage II (4.4% compared with 5.1%; P = 1.000), death (3.7% compared with 3.6%; P = 1.000)
(power: 80%)
Nutritional outcomes: feeding intolerance (at least one episode), 222.9% compared with 48.1% (P , 0.001). TFEF:
11.7 d (10.9812.58 d) compared with 13.2 d (11.1415.4 d) (95% CI: 23.86, 20.87 d); weight gain (g): at 14 d, 1202
(1154.51249.5) compared with 1186 (1137.11234.9) (95% CI: 254.72, 287.09); at 28 d, 1369 (1314.61423.7) compared
with 1378 (1323.51433.9) (95% CI: 299.09, 280.10); and at 42 d, 1571 (1503.41639.8) compared with 1555 (14931617.6)
(95% CI: 2109.19, 2141.72)
n = 150 (study: n = 75 compared with placebo: n = 75). Intervention: (L.acidophilus + L. rhamnosus + L. casei + Lactobacillus
plantarum + B. infantis + S. thermophiles) compared with no probiotics
Type of milk: EBM/formula. Type of delivery: data not available
Primary outcome: development of NEC (stages IIAIIIB), mortality, and combined NEC and death (power: 80%)
Nutritional outcomes [median (range)]: TFEF, 18 d (056 d) compared with 15 d (039 d) (P = 0.092). Total hospital stay: 45
d (13134 d) compared with 40 d (8120 d) (P = 0.343)
n = 31 (study: n = 15 compared with controls: n = 16). Intervention: L. rhamnosus GG compared with no probiotics
Type of milk: EBM. Type of delivery: CS, 47% (study group) compared with 63% (controls)
Primary outcome: effect of enteral probiotics on intestinal blood flow (superior mesenteric artery); P = 0.035 (power 92.3%)
Nutritional outcomes: mean (6SD) TFEF: 23.9 6 8.3 compared with 22.1 6 8.5 d P = 0.55
n = 1099 (probiotic: n = 548 compared with placebo: n = 551). Intervention: (B. infantis + S. thermophilus + B. lactis) compared
with maltodextrin
Type of milk: EBM/formula. Type of delivery: CS, 65.5% (study group) compared with 68.4% (controls)
Primary outcome: LOS (power 80%)
Secondary outcomes: incidence of definite or clinical sepsis, composite outcome of definite or clinical sepsis, number of courses
and duration of antibiotic treatment, incidence of definite sepsis, mortality, incidence of NEC, and at least stage NEC, PDA,
IVH grade 3 or 4, cystic PVL, ROP of at least stage 3, oxygen requirement at 36 wk PMA, or at 28 d of age
Nutritional outcomes: study compared with controls for all outcomes; median (IQR) duration of hospitalization, 71 d (5492 d)
compared with 74 d (5893 d) (P = 0.09); TFEF, 12 d (916 d) compared with 12 d (1017 d) (P = 0.31); mean (6SD) time to
regain birth weight, 11.1 6 4.5 compared with 11.7 6 4.8 d (P = 0.06); weight at 28 d of age, 1495.0 6 401.2 compared with
1446.0 6 379.2 g (P = 0.04); and weight at discharge: 2870.5 6 748.8 compared with 2864 6 738.9 g (P = 0.89)
n = 208 (probiotic: n = 104 compared with placebo: n = 104). Intervention: S. boulardii compared with distilled water
Type of milk: EBM/formula. Type of delivery: CS, 80.8% (study group) compared with 88.5% (controls)
Primary outcome: at least stage II NEC (6.7% compared with 6.7%; P = 1.0), at least stage II NEC or late-onset culture-proven sepsis
(28.8% compared with 23%, P = 0.34), and at least stage II NEC or death (9.6% compared with 7.7%; P = 0.62) (power: 80%)
Nutritional outcomes: mean (6SD) TFEF, 11 6 7 compared with 12 6 7 d (P = 0.37); weight gain (g/wk), 113 6 61 compared
with 129 6 65 (P = 0.31); and median (IQR) duration of hospital stay: 39 d (2860 d) compared with 43 d (2960 d) (P = 0.62)
Other secondary outcomes: oxygen dependency at 36 wk (P = 0.82) and mortality until hospital discharge (P = 0.74)
n = 400 (probiotic: n = 200 compared with placebo: n = 200). Intervention: L. reuteri (DSM 17938) compared with oil-based placebo
Type of milk: EBM/formula. Type of delivery: CS, 75% (study group) compared with 76% (placebo group)
Primary outcome: frequency of NEC and or death after 7 d of life (power 80%), frequency of proven sepsis, rates of feed
intolerance, and duration of hospital stay (power: 80%)
Nutritional outcomes: study compared with placebo for all outcomes; feed intolerance, 28% compared with 39.5% (P = 0.015);
median (range) duration of hospital stay, 38 d (10131 d) compared with 46 d (10180 d) (P = 0.022); and mean (6SD) TFEF,
9.1 6 3.2 compared with 10.1 6 4.3 d (P = 0.006)
n = 159 (study: n = 79 compared with placebo: n = 80). Intervention: B. breve M16 V compared with dextrin
Type of milk: EBM. Type of delivery: CS, 75% (study group) compared with 65% (placebo group)
Primary outcome: B. breve fecal counts (power: 90%)
Nutritional outcome: TFEF [median (IQR)]: 12 d (921, 571 d) for study group compared with 12 d (816, 381 d) for placebo
group; P = 0.306
Secondary outcomes: proven EOS, 5% compared with 3% (probiotics compared with placebo) (P = 0.681); proven LOS,
78% compared with 84% (probiotics compared with placebo) (P = 0.465)
n = 112 (study: n = 56 compared with placebo: n = 56). Intervention: (B. longum + L. acidophilus + B. lactis +
B. bifidum) compared with sterile water
Type of milk: EBM. Type of delivery: CS, 83.9% (study group) compared with 76.8% (controls)
Primary outcome: enteric fungal colonization (overall P = 0.03; for ,1000 g, P = 0.02) (power: 80%)
Secondary outcomes: incidence of LOS (gram positive: 16.1% compared with 32.1%, P = 0.055; gram negative: 21.4% compared
with 26.8%; P = 0.053), NEC (3.6% compared with 3.6%; P = 1.0), and mortality (15.2% compared with 17.21%; P = 0.5)
Nutritional outcomes (means 6 SDs): duration of hospitalization [25.77 6 9.16 compared with 31.21 6 12.67 d (P = 0.002 especially
for ,1000 g); 28.78 6 9.16 compared with 34.21 6 11.68 d; P = 0.004], TFEF [overall, 11.22 6 5.04 d in study group compared with
15.41 6 8.07 d in placebo group (P = 0.016); and in ,1000 g, TFEF of 13.22 6 5.04 compared with 17.41 6 8.07 d (P = 0.014)]
(Continued)

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20) Jacobs, 2013 (58)

Study characteristics

PROBIOTICS AND NUTRITION IN PRETERM NEONATES

1513

TABLE 1 (Continued )
(No. of study) first author,
year of publication
(reference)
25) Totsu, 2014 (63)

Study characteristics
n = 283 (probiotic: n = 153 compared with placebo: n = 130). Intervention: B. bifidum compared with dextrin
Type of milk: EBM/formula. Type of delivery: CS, 50.5% (study group) compared with 79.2% (placebo group)
Primary outcome (mean 6 SD): postnatal day when enteral feed exceeding 100 mL $ kg21 $ d21 (11 6 3.6 d in probiotic group
compared with 12.1 6 3.8 d in controls; P , 0.05) (power: 80%)
Secondary outcomes: morbidities, probiotics compared with controls (LOS: 3.9% compared with 10%; P , 0.05); mean (6SD)
length of hospital stay, 92.3 6 44.5 compared with 92.9 6 40.2 d (P = NS); weight gain in hospital stay (g/d), 20.1 6 3.7
compared with 20.8 6 4 (P = NS); and somatic growth before discharge (head circumference at discharge; P = NS)

For all outcome comparisons, results in the study group are presented first. BBG, Bifidobacterium breve YIT4010; CS, cesarean delivery; EBM,
expressed breast milk; EDD, expected date of delivery; EOS, early-onset sepsis; IVH, intraventricular hemorrhage; LGG, Lactobacillus rhamnosus GG
(ATCC 53103) Gorbach and Goldin; LOS, late-onset sepsis; LS, lower segment; LSCS, lower-segment cesarean section; NEC, necrotizing enterocolitis; PDA,
patent ductus arteriosus; PDHM, pasteurized donor human milk; PMA, postmenstrual age; PVL, periventricular leukomalacia; ROP, retinopathy of prematurity; TFEF, time to full enteral feed; VD, vaginal delivery.
2
Study numbers 17A, 17B, and 17C are 3 publications from the same study population.

5) Incomplete outcome data: The ROB in this domain was

assessed as low if all or a majority ($80%) of participants
contributed to outcome data. If some outcome data were
missing, but reasons were reported, and balanced across
groups, the assessment was considered low ROB. The risk
was assessed as high if this was not the case. The risk was
assessed as unclear when this information was not given in
a clear way.
6) Selective reporting of outcomes: The ROB in this domain
was assessed as low if all outcomes prespecified in methodology were reported. The risk was assessed as high if
some of the prespecified outcomes were missing in the
results section. The risk was assessed as unclear when this
information was not given in a clear way.
7) Other sources of bias: The ROB in this domain was assessed as low or high based on absence or presence of any
other sources of bias, as per the reviewers judgment.
Differences of opinion were resolved by consensus after a
group discussion involving all authors.
Data synthesis and statistical analysis
The meta-analysis was performed with Review Manager
software (version 5.2; Cochrane Collaboration, Nordic Cochrane
Centre). The random-effects model was used because we anticipated clinical and statistical heterogeneity (26). For data that
were not suitable for the meta-analysis, results have been provided in table format. Mean differences and 95% CIs were
calculated for continuous variables. For binary outcomes, we
planned to use RRs and 95% CIs.

the neonatal period (35). Therefore, we aimed to conduct the following 3 subgroup analyses: Bifidobacterium or non-Bifidobacterium
strains, single- or multiple-strain usage, and early (#72 h) or late
(.72 h) initiation of probiotic supplementation. Previous systematic reviews have not addressed these important issues.
Heterogeneity and assessment of publication bias
Clinical heterogeneity was assessed and reported by summarizing characteristics such as the study population, type, dose and
duration of probiotic supplementation, and other characteristics.
Statistical heterogeneity was estimated by using the I2 statistic.
Publication bias was assessed by using the funnel plot (36).
RESULTS

Of 466 citations that met criteria after the initial broad database
search, 25 studies (27 publications) were included in the review
(Figure 1; 3763). There were 3 publications from the same
study population by Demirel et al. (5355) that evaluated the
effect of probiotic supplementation on different outcomes;
hence, we have considered these 3 publications as a single study.
The total sample size in the review was 5895 subjects with
a median of 183 subjects (range: 311099 subjects). A total of
14 trials used a placebo (n = 3812). Fifteen trials used singlestrain probiotic supplementation. Only 2 trials [Samanta et al.
(43) and Totsu et al. (63)] reported the TFEF as the primary
outcome. Only one trial [Al Hosni et al. (48)] reported extrauterine growth restriction as the primary outcome. Other
trials assessed NEC, sepsis, mortality, gut colonization, gut
permeability, and other outcomes as major outcomes. Details
of study participants, sample sizes, interventions, and outcomes of included RCTs are given in Table 1.
Primary outcome

Prespecified subgroups
Bifidobacterium are the dominant beneficial gut flora in healthy
breast-milkfed infants (32). Evidence has indicated that supplementation with multiple strains may be more effective than with a
single strain (33, 34). Investigators have also reported better colonization of the gut if supplementation is commenced very early in

A total of 19 of 25 trials were included for the meta-analysis

for the outcome of the TFEF (Table 1). Overall, the probioticsupplemented group took less time to achieve full feeds (mean
difference: 21.54 d; 95% CI: 22.75, 20.32 d; P 0.01; I2
93%). This improvement was noted irrespective of whether
Bifidobacterium or non-Bifidobacterium strains (Figure 2) or

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outcome assessors were blinded. The risk was assessed as

high if they were not blinded. The risk was assessed as
unclear when this information was not given in a clear way.

1514

ATHALYE-JAPE ET AL.

whether single or multiple strains were used (Figure 3). Beneficial effects were also shown in both early and late commencements of probiotics (Figure 4).
Secondary outcomes
Secondary nutritional outcomes reported in trials are reported
in Table 1. Probiotic supplementation was shown to reduce the
duration of hospitalization (43, 50, 60, 62), incidence of feed
intolerance (37, 46, 50, 55, 60), and duration of indirect
hyperbilirubinemia (53). The supplementation improved weight
gain in the hospital (37, 46) and growth velocity (48). None of the
trials showed any adverse effects of probiotics on these or other
defined outcomes.
ROB
Results of the ROB assessment are reported in Table 2. All but
one trial used some form of a random sequence generation
method. Allocation concealment was achieved in 17 of 25 trials
but was unclear in the remaining trials. The funnel plot for the
assessment of publication bias showed a symmetrical distribution of most studies (Figure 5).
DISCUSSION

Our systematic review (25 trials; 5895 preterm neonates)

indicated that probiotic supplementation has beneficial effects on

enteral nutritionrelated outcomes. The TFEF (19 trials; 4527

neonates) was reduced significantly in the probiotic supplemented group. This improvement was noted irrespective of
whether Bifidobacterium or non-Bifidobacterium strains or
whether single or multiple strains were used. Beneficial effects
were also shown in both early and late commencements of
probiotics. Other benefits included increased weight gain and
a reduced time to regain birth weight, reduced duration of
hospital stay, and fewer episodes of feed intolerance.
Probiotics have the potential to improve gut maturity and the
function by various pathways. Abrams and Bishop (69) first
reported the influence of microorganisms on intestinal motility.
They observed that both small and large intestinal transit times
and gastric emptying decreased in germ-free animals. Verdu et al.
(70) reported that Lactobacillus rhamnosus and Lactobacillus
helvaticus supplementation normalized gastric functioning in rat
models. Williams et al. (71) showed that commensals in the gut
as well as probiotics improved gut motility. Indrio et al. (72)
showed that infants who received Lactobacillus reuteri DSM
(Deutsche Sammlung von Mikroorganismen) 17938 had shorter
gastric emptying times than did infant who received a placebo.
Indrio et al. (73) have also reported that L. reuteri DSM 17938
increased the proportion of electrical activity that turned into
peristaltic movements in infants. Probiotics modulate gut motility possibly by their secretory products or end products of
fermentation by influencing intestinal neuroendocrine factors
and the influence of mediators secreted by the gut as an immune
reaction (74). Short-chain fatty acids (SCFAs) are the main end

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FIGURE 2 Probiotics compared with controls: subgroup analysis by presence or absence of Bifidobacteria. Fern. -Carro., Fernandez-Carrocera; IV,
inverse variance.

PROBIOTICS AND NUTRITION IN PRETERM NEONATES

1515

products of colonic fermentation by gut microbiota of dietary

fiber. SCFAs contribute to the regulation of upper gastrointestinal
motility (lower esophageal sphincter and gastric relaxation),
a reduction in the gastric-emptying time, lower interdigestive acid
output, and overall gut homeostasis (75). Mechanisms of action of
SCFAs on gut motility involve systemic, humoral, and neural
pathways, local reflexes, and myogenic responses (76). Butyrate
plays an important role in the regulation of transepithelial fluid
transport, maintaining epithelial integrity, ameliorating mucosal
oxidative status, and inflammation and modulating gut motility
and visceral sensitivity (77, 78). Evidence from a small, randomized, double-blind, placebo-controlled trial indicated that
probiotic supplementation significantly improved the postprandial
superior mesenteric artery flow (P = 0.035) (57), which is another
possible pathway by which probiotics may improve feed tolerance because a rise in the postprandial mesenteric flow is essential
for food digestion (57).
In a systematic review of adult literature, Chapman et al. (33)
examined whether multiple-strain probiotics are better than
single strain in improving health outcomes. The authors showed
that, in 12 of 16 included studies, mixtures of probiotics had
better beneficial effects than did single strains in irritable bowel
syndrome, diarrhea, atopic disease, gut function, immune
function, respiratory tract infections, gut microbiota modulation,
inflammatory bowel disease, and Helicobacter pylori infection.
Chapman et al. (33) suggested that synergistic effects of various
strains may contribute to such beneficial effects. In our study, the

use of multiple-strain probiotics decreased the TFEF by 1.74 d

compared with that with no supplementation, whereas the use of
single strains reduced the TFEF by 1.34 d. Additional RCTs are
needed to address this issue in the preterm neonatal population.
Our meta-analysis showed that full enteral feeds were achieved
3.43 d sooner in the late-supplementation group than controls. In
contrast, benefits were less pronounced in the early supplementation group (1.16 d). Even after a careful review of factors
such as gestation, birth weight, type, dose, and duration of
supplementation, and other factors, we were unable to identify
reasons behind such better benefits with late supplementation.
Results from any subgroup analyses should not be overinterpreted.
Unless there is strong supporting evidence, the results are best
viewed as a hypothesis-generation exercise (79). Hence, additional RCTs are essential in which early commencement is directly compared with late probiotic supplementation.
The results of one such RCT were reported by Yamasaki et al.
(35). Thirty-six very-low-birth-weight infants were randomly
assigned to early (,48 h after birth) and late (.48 h after birth)
supplementation with Bifidobacterium bifidum. The median
(range) time to reach a feeding volume of 100 mL (per kg/d)
was 10 d (713 d) in the early group and 11 d (1015 d) in the
late-supplementation group. The daily body weight gain was
significantly higher in the early supplementation group (21.4 6
3.2 g/d compared with 18.3 6 4.0 g/d; P , 0.02). The highest
colonization rate of Bifidobacterium was observed when the supplementation was started between 24 and 48 h after birth (35).

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FIGURE 3 Probiotics compared with controls: subgroup analysis by number of strains (single or multiple). Fern. -Carro., Fernandez-Carrocera; IV,
inverse variance.

1516

ATHALYE-JAPE ET AL.

Different probiotic strains may have effects on different aspects of gut maturity and function and to different extents. Hence,
large definitive trials are needed to assess specific strains for the
optimization specific enteral nutrition-related primary outcomes
in preterm neonates. However, because it has been proven beyond
doubt that probiotic supplementation reduces risk of death and
NEC, placebo controlled trials to evaluate nutritional outcomes in
which the control arm receives no probiotics are unethical (26).
Strains with a documented ability to stimulate gut motility and
gastric emptying and reduce the incidence and severity of NEC
such as L. reuteri DSM 17938 may be suitable for such trials
(11, 8084). For example, future RCTs could compare probiotics
with L. reuteri DSM 17938 with probiotics without L. reuteri
DSM 17938.
For primary outcomes such as postnatal growth restriction, it is
important to use standardized parenteral and enteral nutrition
protocols in the trial protocol. With consideration of direct and
indirect clinical and economic benefits of a reduced duration of
hospital stay and parenteral nutrition, an assessment of economic
benefits and long-term neurodevelopment is important.
Strengths of our study were the large sample size (n = 5895)
and the fact that significant benefits were observed irrespective
of the variations in patient characteristics and the probiotic
strain, dose, time of commencement, and duration of supplementation. Unlike a randomized trial, which usually involves the
comparison of 2 specific treatment strategies inn a defined
population, the meta-analysis enabled the combination of trials
with similar conceptual intents but quite different treatments

(85). For example, in a breast cancer meta-analysis in which trials

with different chemotherapy combinations, doses, and durations
were combined, results confirmed that polychemotherapy was
effective in reducing mortality and the recurrence of cancer (86).
Study limitations were as follows: 1) nutrition-related effects
(e.g., TFEF) were not the primary outcomes in a majority of
RCTs (23 of 25 RCTs); and 2) there was significant statistical
heterogeneity (I2 = 93%).
Sample sizes in RCTs were calculated to ensure that the trial
had adequate power to answer a query pertaining to the primary
outcome of interest. Hence, a meta-analysis of secondary outcomes needs to be interpreted cautiously. Statistical heterogeneity examines whether the observed variability of effects is
greater than that expected by chance alone (87). We explored
heterogeneity by conducting various sensitivity analyses (e.g., by
excluding studies in which a placebo was not used and allocation
concealment was not adequate. However, statistical heterogeneity continued to persist.
In conclusion, our results indicate that probiotic supplementation reduces TFEFs in preterm neonates. Such benefits and other
nutritional outcomes such as weight gain, growth velocity, and the
duration of hospital stay need to be explored in future RCTs.
In addition, benefits of strategies such as single- compared with
multiple-strain probiotics and early compared with late commencements of probiotics need to be tested in future trials.
This work has not been published previously except in the form of an abstract at the 2014 annual meetings of the Perinatal Society of Australia and

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FIGURE 4 Probiotics compared with controls: subgroup analysis by time of commencement (early or late). Fern. -Carro., Fernandez-Carrocera; IV,
inverse variance.

1517

PROBIOTICS AND NUTRITION IN PRETERM NEONATES

TABLE 2
Risk of bias assessment in included studies
First author, year of
publication (reference)

Allocation
concealment

Blinding of
participants
and personnel

Blinding
of outcome
assessment

Incomplete
outcome data

Selective
reporting

Free of
other bias

Unclear
Unclear
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Unclear
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Unclear
Yes
Yes
Yes
Yes

Unclear
Unclear
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Unclear
Yes
Unclear
Yes
Yes
Yes
Unclear
Yes
Unclear
Yes
Unclear
Unclear
Yes
Yes
Unclear

Yes
Yes
Yes
Unclear
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Unclear
Yes
Unclear
Yes
Yes
Yes
Yes
Yes
Unclear
Yes
Unclear
Unclear
Yes
Yes
Yes

Yes
Unclear
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Unclear
No
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Unclear
Yes
Yes
Unclear
Unclear
Yes

Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
Yes
Yes

No
Yes
No
Unclear
Unclear
No
No
No
No
Unclear
Unclear
No
Yes
No
No
No
No
No
No
No
No
Unclear
Unclear
No
No
No
Unclear

Yes
Unclear
Yes
Unclear
Yes
Yes
Yes
Yes
Yes
Yes
Unclear
Yes
Unclear
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Unclear
Yes
Unclear
Yes
Yes

New Zealand in Perth, Western Australia, and the Pediatric Academic Society
of the United States in Vancouver, Canada.
The authors responsibilities were as followsGA-J: independent literature search and writing of the first and final drafts of the manuscript; SR and
SP: supervision of GA-J; GD: independent literature search, rechecking and
interpretation of data, and writing of the first and final draft of the manuscript; SR: rechecking literature search results and data, handling the metaanalysis software, and supervising the first and final draft of the manuscript;
SP: concept and design, rechecking and interpreting data, and supervising of
the first and final drafts of the manuscript; and all authors: saw and approved
the final version of the manuscript that was submitted. None of the authors
declared a conflict of interest.

FIGURE 5 Funnel plot to assess publication bias. MD, mean difference.

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