Do Probiotics Reduce The Risk Of Severe Necrotising Enterocolitis (NEC) In Infants Born Before 32 Weeks Gestation? An Observational Study Using Routinely Collected Data.
Conditions: Enterocolitis, Necrotizing
Sponsor: Imperial College London

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EVALUATING THE EFFECT OF PROBIOTICS ON SEVERE NECROTISING 
ENTEROCOLITIS IN PRETERM INFANTS BORN BEFORE 32 WEEKS GESTATION: A 
PROPENSITY-MATCHED POPULATION STUDY  
 
Alice Aveline1, Lisa Szatkowski 2, Janet Berrington3, Kate Costeloe4, Shalini Ojha5, Paul Fleming4 and 
Cheryl Battersby1 
1. Neonatal Medicine Research Group, Imperial College London, UK and Centre for Paediatrics and 
Child Health, Imperial College, London, UK  
2. Centre for Perinatal Research, Lifespan and Population Health, School of Medicine, University of 
Nottingham, UK 
3. Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne, UK and 
Newcastle Upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK 
4. Queen Mary, University of London, UK and Homerton Healthcare NHS Foundation Trust, London, 
UK 
5. Centre for Perinatal Research, Lifespan and Population Health, School of Medicine, University of 
Nottingham, UK and Neonatal Unit, University Hospitals of Derby and Burton NHS Trust, Derby, UK  
 
 
 
 
 
Correspondence to Cheryl Battersby c.battersby@imperial.ac.uk  
 
 

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ABSTRACT 
Background  
Necrotising enterocolitis (NEC) is a leading cause of mortality and morbidity in preterm infants. 
Whilst there have been numerous clinical trials, there remains continued uncertainty  concerning 
the benefits of probiotics and the different strains [1]. We aim to compare the effect of early 
probiotic  exposure (within 14 postnatal days) versus no (or ≥14 days) probiotic exposure on odds of 
NEC. 
Methods  
We will conduct a observational, propensity score matched study using data from the UK National 
Neonatal Research Database (NNRD). Infants born <32 weeks gestation cared for in neonatal units in 
England and Wales between 01/01/2016 and 31/12/2022 will be included.  We will exclude infants 
who died before day four of life and those with major congenital anomalies. A propensity score 
matched approach will be applied, matching for two critical variables gestational age and birth year 
epoch, and an additional 17 variables to control for background characteristics and initial severity of 
illness. We will compare outcomes between  infants exposed and not exposed to probiotics in the first 
14 days. The primary outcome will be severe NEC (confirmed surgically, by postmortem or a cause of 
death).  Sensitivity analyses, including analysing the whole cohort are planned to assess robustness of 
the results.  
 
KEY WORDS 
Probiotics, preterm infants, necrotising enterocolitis, retrospective cohort studies, population study, 
propensity score, England, United Kingdom 
 
 

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INTRODUCTION 
Necrotising Enterocolitis (NEC) remains one of the most important causes of morbidity and mortality 
of preterm infants for which there is no widely agreed reliably effective preventative intervention. 
While maternal breast milk has been shown to reduce the risk of NEC, the majority of NEC occur in 
exclusively maternal milk fed infants [2-4] . Over the past several decades the possibility that 
probiotics, live organisms consumed orally into the gut, might safely prevent NEC has been 
investigated in an extensive series of randomised controlled trials and observational studies examining 
different probiotic strains. The interpretation of these studies has been controversial and has resulted 
in a potentially confusing range of advice to clinicians.    
The  largest  randomised controlled trials (RCT) were powered to examine the primary outcome of 
late-onset-sepsis, and were underpowered to examine necrotising enterocolitis [3, 4]. When 
individual studies were meta-analysed in a Cochrane review in 2023, probiotics were shown to reduce 
the risk of NEC (RR 0.54, 95% CI 0.46-0.65; I² = 17%; 57 trials, 10,918 infants; low certainty); probably 
reduce mortality (RR 0.77, 95% CI 0.66-0.90; I² = 0%; 54 trials, 10,484 infants; moderate certainty); 
and have little or no effect on the risk of late‐onset invasive infection (RR 0.89, 95% CI 0.82-0.97; I² = 
22%; 49 trials, 9876 infants; moderate certainty).  Whilst data for extremely low birthweight (<1kg) 
infants were limited, the authors concluded that probiotics may have little or no effect on NEC (RR 
0.92, 95% CI 0.69- 1.22, I² = 0%; 10 trials, 1836 infants; low certainty), all‐cause mortality (RR 0.92, 
95% CI 0.72- 1.18; I² = 0%; 7 trials, 1723 infants; low certainty), or late‐onset invasive infection (RR 
0.93, 95% CI 0.78-1.09; I² = 0%; 7 trials, 1533 infants; low certainty) in this group.  The review 
recommends further large randomised controlled trials to provide evidence of sufficient validity and 
applicability to inform policy and practice [1].     
Recommendations from professional bodies vary globally.  The European Society for Paediatric 
Gastroenterology Hepatology and Nutrition (ESPGHAN) made conditional recommendations in 2020, 
that certain probiotic strains and probiotic combinations could be used as potential preventative 
strategies for NEC [5]. In 2021 the American Academy of Pediatrics issued a statement not supporting 
routine universal probiotic administration, especially to infants <1000g, citing a lack of evidence of 
benefit in modern trials and limited availability of suitable pharmaceutical grade probiotics (in the 
United States) [6].  More recently, following concerns about links to cases of sepsis, the US Federal 
Drug Administration (FDA) has issued warning letters to companies manufacturing probiotics 
for illegally selling probiotic products to treat or prevent diseases in preterm infants in the absence of 
FDA approval for this use [7]. 

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Probiotic use in United Kingdom neonatal units has increased significantly over the past six years, with 
a survey conducted in 2022 showing that around 44% (70/161) of responding units in year 2022 
routinely use probiotics [8].  The two most commonly used probiotics in the UK are a combination of 
Bifidobacterium infantis, Streptococcus thermophilus and Bifidobacterium lactis (trade name 
PROPREMS abbreviated as PP) and a product containing Lactobacillus acidophilus, Bifidobacterium 
bifidum and Bifidobacterium longum spp infantis (trade named Labinic, abbreviated as LB) [8]. Infant-
level descriptive data from England and Wales confirmed the rising use of probiotics over time such 
that half of infants of infants born in year 2022 born less than 32 weeks were exposed to probiotics 
(unpublished work).  
The UK-based National Neonatal Research Database (NNRD) includes data from all infants admitted 
to neonatal units in England and Wales (approximately 8,000 of the 100,000 admissions per year are 
preterm infants born <32 weeks gestation) and provides an opportunity to assess the impact of an 
intervention on specific neonatal outcomes using real world data.   
In this study we aim to apply causal inference methods, including propensity matching to NNRD data 
to determine whether probiotic use is associated with a reduction in severe NEC in infants born < 32 
weeks gestation.   
METHOD 
Study design 
This retrospective cohort study will use existing, routinely collected data held in the NNRD.  
Data source 
The NNRD contains records for all infants admitted to neonatal units in England and Wales since 2012 
[9]. Relevant variables will be extracted from the NNRD. These included patient demographics, 
baseline comorbidities, diagnoses, elements of intensive care and outcomes.  
Study population 
Inclusion criteria: We will include infants born 23+0 to 31+6 weeks completed gestation between 
January 1st 2016 and December 31st 2022 (7 years) admitted to neonatal units in England and Wales.  
Exclusion criteria: We will exclude infants with: 
• 
Data missing for any of gestational age at birth, birth weight and birth year 

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• 
Data missing from the first admission to neonatal care following birth (first episode of care) 
or where data only exists from  day 3 of life onwards  
• 
birth weight for gestational age z score exceeds 4  
• 
Died within the first four days of life 
• 
Major congenital abnormality (Supplementary Material 1 ) 
Definitions 
Exposure 
Exposed to probiotics: Documented receipt of any probiotic in the first 14 days of life. 
Not exposed to probiotics  (Comparator): No documented receipt of a probiotic in the first 14 days of 
life.  
Primary outcome 
Severe NEC defined as NEC confirmed at surgery or postmortem or NEC included as a cause of death 
in the NNRD [10]. (Supplementary Material 2) 
Key secondary outcomes  
▪ 
Alternative NEC definitions (Supplementary Material 3 for detail of how these NEC definitions will 
be derived from variables available in the NNRD). 
o National Neonatal Audit Programme (NNAP) definition [11] i.e. NEC diagnosed at 
postmortem or during surgery or using clinical and radiographic features.   
o Pragmatically defined NEC i.e. recorded diagnosis of NEC and received at least 5 
consecutive days of antibiotics whilst nil by mouth. 
▪ 
Late onset sepsis - Either a positive blood culture report of the growth of any organism from the 
NNAP late onset infection list of “Clearly pathogenic organisms” OR “other organisms” which 
includes coagulase negative Staphylococcus [11] OR a discharge diagnosis indicating one or more  
“clearly” or “other” pathogenic organisms from the NNAP lists (Supplementary Material 4).  
▪ 
Survival to discharge from neonatal care 
Other secondary outcomes  
These include severe brain injury treated retinopathy of prematurity (ROP), bronchopulmonary 
dysplasia (BPD) and severe BPD  (definitions in Supplementary Material 3) together with the 

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composite outcomes; survival without severe NEC, survival without severe NEC or late onset sepsis, 
survival without any NEC and time to full feeds . 
 
Statistical analysis 
Sample size and power calculation 
The reduction in risk of NEC in exposed infants is of the order of 0.54 compared to those not 
exposed [1], and we hypothesise a similar reduction in severe NEC. The estimated UK incidence of 
severe NEC in this population is approximately 3.1% [2]; we therefore expect the risk in the exposed 
group to be 1.7%. Assuming two-sided alpha of 0.5%, matching 7,000 each of exposed and non-
exposed infants would detect a difference with 99% power.  
Descriptive analyses 
We will report the proportion of our cohort exposed to probiotics for each year in the study period.  
We will also report median postnatal day when probiotics are commenced.  
Missing data 
We will examine rates of missingness for the exposure, outcome and all covariates. To determine 
whether multiple imputation is required, we will examine whether missingness of any covariate is 
associated with the exposure and outcome and whether the covariate value is significantly associated 
with the exposure and outcome.  
Creating the matched sample 
To address the issue of confounding, our primary analysis will use a propensity matching methodology. 
This will be divided into two stages; first we will estimate the probability that each infant would be 
exposed to probiotics (a propensity score) and second, we will match exposed infants to unexposed 
infants with similar propensity scores.  
To create the propensity score, we first identified factors that influence probiotic receipt and risk of 
severe NEC by creating a directed acyclic graph (DAG)  (Figure 1), informed by review of the scientific 
literature and pooled experience of the authors. We next mapped concepts in the DAG to related 
variables within the NNRD where available (Table 1). Finally, propensity scores will be created for each 
infant, using a logistic regression model (the propensity score model) where probiotic exposure is the 
dependent variable and the NNRD variables are the independent variables.  
To determine which NNRD variables should be included in the propensity score model, we will assign 

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a level of importance (critically, highly or moderately important) to each variable. Critically and highly 
important variables will be mandatory in the propensity score model. The step-wise method described 
by Imbens and Rubins will be used to select the moderately important variables that are incorporated 
in the propensity score model [12] (more details in Supplementary Material 5). Critically important 
variables included gestational age and birth year [13]. Highly important variables include precise 
gestation (in days), birthweight, network, level of care on day 3 of life, sex and year of birth. 
Moderately important variables are shown in Table 1. 
After creating the propensity scores, we will create a matched sample within strata defined by the 
two critically important variables, gestational age and birth year group. The groups are GA: 22+0 to 
24+6 weeks, 25+0 to 27+6 weeks, 28+0 to 29+6 weeks, 30+0 to 31+6 weeks and birth year: 2016-2019 
or 2020-2022, resulting in eight groups in total. Within each strata, we will match each exposed infant 
to an unexposed infant with a similar propensity score. We will use nearest neighbour matching with 
a caliper of 0.2 standard deviations. As the nearest neighbor matching method matches infants in a 
particular order; that order can affect the quality of both individual matches and the matched 
sample. We will therefore repeat the matching process twenty times, matching infants in a random 
order each time.  
To test the association between probiotic exposure and outcomes, logistic regression will be applied 
to the matched sample to assess the outcomes in exposed and unexposed infants. Robust standard 
errors will be used to correct for correlation within a NNU and within infants born to the same mother. 
Bonferroni corrections will be applied to analyses of the secondary outcomes. 
Mortality over the first 28 days after birth will be assessed using a log rank test and presented with 
Kaplan Meier curves. 
Examining the incidence of NEC by probiotic strain  
We will measure the association between the type of probiotic (LB or PP in the first 14 days of life) 
and odds of severe NEC. Given the smaller sample size of this subset of infants, we will use 
multivariable logistic regression rather than propensity matching. We will adjust for variables that are 
included in the propensity score analysis and will use robust standard errors in the logistic regression.  
Whilst LB has been used in UK neonatal units since 2016, PP was only used at scale in the UK from mid 
2020. To avoid confounding secular and strain influences, we will conduct a sensitivity analysis 
restricting the analysis to the period June 2020 to Dec 2022. 
Subgroup analyses 

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We will conduct a pre-planned subgroup analysis for infants born < 28 weeks vs born ≥ 28 weeks to 
explore the treatment effect within specific populations for the primary outcome, severe NEC, and 
key secondary outcomes (NNAP defined NEC, pragmatically defined NEC, late onset sepsis and 
survival to discharge). To limit the risk of type 1 errors, we will not conduct subgroup analyses for 
the other secondary outcomes and Bonferroni corrections will be applied to the analyses of the 
secondary outcomes.  
In addition, we will conduct a subgroup analysis for infants born weighing < 1kg vs ≥ 1kg. Given the 
substantial anticipated overlap between the populations in the gestational age subgroups and the 
birthweight subgroups, we will restrict this analysis to the primary outcome.  
Sensitivity analyses 
Whole cohort: Logistic regression without propensity matching  
We will conduct a standard logistic regression analysis on the whole cohort without propensity 
matching to evaluate whether the findings differ. This analysis will be adjusted for all the variables 
included in the propensity score model for the matched sample. 
Unit-level comparisons 
To take account of potential cross-contamination of probiotics between infants cared for in the 
same NICU, we will repeat our analysis with the assumption that all infants cared for in a “probiotic 
unit” are exposed to probiotics regardless of the infant-level exposure status.  
A probiotic unit is defined as one where:  
1. Over 50% of cohort infants born in the same month, who spent day 3 of life on the unit, were 
exposed to probiotics in first 14 days of life OR  
2. At the time the infant was born, the unit had implemented a protocol for routine use of probiotics 
for infants born < 32 weeks gestation. Information about protocols for probiotics was ascertained 
using a survey.    
We will restrict this analysis to tertiary Neonatal Intensive Care Units (NICU; designation as at July 
2023) because the smaller number of infants <32w in non-tertiary units may result in 
misclassification of a unit’s probiotic status.  
Because of the potential for type 1 error due to multiple comparisons, treatment effects estimated 
with sensitivity analyses and in subgroup analyses will be interpreted as exploratory.  
Ethics 

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Ethics approval was granted by the South-East Scotland Research Ethics Committee 01 (REC 
reference 23/SS/0016) for use of the NNRD data as part of a larger study evaluating the impact of 
introduction of a care bundle on incidence of NEC [14].  
Table 1: Concepts from the directed acyclic graph and the NNRD variables which will be used to 
operationalise those concepts 
Concept from 
DAG  
NNRD variable used to 
operationalise the concept 
Operationalised in propensity score  
Probiotics 
DrugsDay 
Binary. Coded as exposed to 
probiotics if NNRD field DrugsDay 
included any one of the following 
terms: Labinic, Proprems, 
Bifidobacterium, Bio-kult, Infloran, 
LB2 in the first 14 days of life. 
Severe necrotising 
enterocolitis 
See Supplementary Materials 2 
Binary. Coded as severe NEC 
present=1, No severe NEC =0 
Gestational age 
GestationWeeks and GestationDays 
Critically important background 
variable. 
 Binary <28 weeks or ≥ 28 weeks  
AND 
Highly important background 
variable. 
 Continuous variable measured in 
days 
Birth year 
BirthYear 
Critically important background 
variable. Categorical: 2016-2018, 
2019-2020 or 2021-2022 
AND 
Highly important background 
variable. 
Categorical: Measured in years 
Birthweight 
Birthweight 
Highly important background 
variable. 
 Continuous measured in grams  
Neonatal Unit  
ProviderNHSCode  
 
Highly important background variable 
(Hospital network on day 3 after birth 
derived from name of neonatal unit 
providing care on day 3). 
 Categorical:  East Midlands; East of 
England; North Central & North East 
London; North West London; North 
West; Northern; South East Coast; 
South London; South West; Thames 
Valley & Wessex; Wales; West 
Midlands; Yorkshire & Humber.   

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BAPM2011 
Highly important background variable 
(Maximum level of care in first 4 days 
of life).  
Categorical: Intensive care, High 
dependency care, Special Care, 
Normal Care  
Sex 
SexPhenotype 
Highly important background variable  
Binary: Male=1, Female=0. 
Ethnicity 
MumEthnicity 
Moderately important background 
variable  
Categorical: White, Mixed, 
Asian/Asian British, Black/Black 
British, Other, Missing. 
Index of Multiple 
Deprivation 
quintile 
PostCodeMotherLSOA 
Moderately important background 
variable  
(Derived from mother’s Lower Level 
Super Output Area).  
Categorical: Most deprived; quintile 2; 
quintile 3; quintile 4; Least deprived.  
Gravidity 
NumberOfPreviousPregnancies 
Moderately important background 
variable  
Categorical: 0, 1, 2-3, 4-5, >5 
Multiple birth 
FetusNumber 
Moderately important background 
variable.  
Categorical: Multiple birth=1; 
Singleton=0. 
Maternal infection MaternalPyrexiaInLabour38c, 
IntrapartumAntibioticsGiven, 
Chorioamnionitis. 
Moderately important background 
variable.  
Binary: Infection=1, No infection=0 
(Coded as 1 if any of 
MaternalPyrexiaInLabour38c, 
IntrapartumAntibioticsGiven, 
Chorioamnionitis are coded as ‘Yes’ in 
NNRD) 
Antenatal steroids 
SteroidsAntenatalGiven 
Moderately important background 
variable.  
Binary: Any antenatal steroids given 
during pregnancy (partial or complete 
course) =1; No antenatal steroids 
given=0.  
Illness severity  
InotropesGiven, DrugsDay, 
RespiratorySupport, NitricOxide 
Moderately important background 
variable. Categorical  
Illness severity score derived from the 
sum of: 
1. Did the infant receive inotropes on 
either day 1 or 2 (coded as 1 or 0) 
(NNRD field: InotropesGiven OR any 
of the following drugs listed in the 
DrugsDay field: Adrenaline, 
Dopamine, Dobutamine, Milrinone, 
Noradrenaline or Vasopressin) 

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Did the infant receive any invasive 
respiratory support on either day 1 or 
2 (coded as 1 or 0) (NNRD fields: 
RespiratorySupport)  
3. Did the infant receive any nitric 
oxide on either day 1 or 2 (coded as 1 
or 0) (NNRD field: NitricOxide) 
Intrauterine 
growth restriction 
(IUGR) 
BW_UKWHO 
Moderately important background 
variable.  
Binary: IUGR=1, No IUGR=0 (Coded as 
1 if birthweight-for-age z-score <-2SD. 
Derived from birthweight z-score 
calculated against reference cohort 
[18]) 
Surfactant 
SurfactantGivenResuscitation, 
DrugsDaily 
Moderately important background 
variable.  
Binary: Received pulmonary 
surfactant = 1, Did not receive 
surfactant = 0 (Coded as 1 if 
surfactant is given either in the 
delivery room or on the neonatal unit 
on the day of birth). 
Enteral feeds 
DayEnteralFeeds 
Moderately important background 
variable.  
Binary: Nil by mouth=1, Fed enterally 
=0 (Coded as 1 if infant was fed 
enterally in the first four days after 
birth). 
Gut ischaemia 
n/a 
Not recorded in NNRD 
Pre-eclampsia 
ProblemsDuringPregnancy 
Moderately important background 
variable.  
Binary: Pre-eclampsia present=1, Pre-
eclampsia absent =0. 
Placental 
abruption 
ProblemsDuringPregnancy 
Moderately important background 
variable.  
Binary: Abruption occurred=1, No 
abruption=0. 
Mode of delivery 
ModeOfDelivery 
Moderately important background 
variable.  
Binary: Vaginal=0; Caesarean 
section=1; Missing. 
Perinatal hypoxia 
MethodsOfResuscitation 
Moderately important background 
variable.  
Binary: Perinatal hypoxia=1; No 
perinatal hypoxia=0 (Coded as 1 if 
infant required significant 
resuscitation at birth, defined as one 
or more of cardiac compressions, 
adrenaline or other drugs. 
 

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Figure 1: Directed acyclic graph summarising critical relationships involved in the association 
between probiotics and severe NEC.  
  
 
 
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FUNDING STATEMENT 
This work was supported by a NIHR Advanced Fellowship awarded to Dr Cheryl Battersby (reference: 
NIHR300617) and by an Imperial College PhD studentship held by Dr Alice Aveline. 
Data extraction costs are covered by NIHR RfPB grant (NIHR203590; Chief Investigator: Shalini Ojha; 
Sponsor: University Hospitals of Derby and Burton NHS Foundation Trust).  
This study was also supported through the Imperial NIHR Biomedical Research Centre. This article is 
independent research funded by the NIHR, and the views expressed in this publication are those of 
the authors and not necessarily those of the NHS, NIHR, or the Department of Health. None of the 
funders have had any influence over study design, collection, analysis and interpretation of the data, 
in writing the report and in the decisions to submit this article for publication. 
 
 

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SUPPLEMENTARY MATERIAL 1: MAJOR CONGENITAL 
ABNORMALITIES   
Major congenital gastrointestinal malformations 
Correction of congenital atresia of oesophagus, oesophageal atresia, oesophageal atresia with distal 
tracheo-oesophageal fistula, oesophageal atresia with tracheoesophageal fistula, oesophageal 
atresia without distal fistula, oesophageal atresia without tracheoesophageal fistula, thoracotomy 
and repair of oesophageal atresia and tracheo-oesophageal fistula with primary anastomosis, atresia 
and stenosis of small intestine, atresia and stenosis of duodenum, duodenal atresia / stenosis / web 
(specify), duodenal atresia / stenosis / web, duodenal atresia / stenosis, duodenal atresia, atresia 
and stenosis of ileum, ileal atresia / stenosis (specify), ileal atresia / stenosis, jejunal atresia / 
stenosis (specify), jejunal atresia / stenosis, atresia and stenosis of large intestine, congenital 
absence atresia / stenosis  parts of large intestine, congenital absence atresia / stenosis parts of 
large intestine, congenital absence atresia / stenosis of rectum with fistula, congenital absence 
atresia / stenosis rectum without fistula, congenital absence atresia / stenosis anus with fistula, 
congenital absence atresia / stenosis anus without fistula, congenital absence atresia / stenosis of 
large intestine part unspecified, atresia of oesophagus without fistula, atresia of oesophagus with 
tracheo-oesophageal fistula (tof), recurrent tracheo-oesophageal fistula, tracheo-oesophageal fistula 
(h-type), congenital tracheo-oesophageal fistula without atresia (tof), congenital stenosis and 
stricture of oesophagus, congenital stenosis of the oesophagus, congenital oesophageal web, 
oesophageal web, large bowel or rectum - atresia, high anorectal anomaly with rectourethral fistula, 
high anorectal anomaly with rectovesical fistula, high anorectal anomaly with rectocutaneous fistula, 
high anorectal anomaly with rectocloacal fistula, high anorectal anomaly with fistula (specify), high 
anorectal anomaly without fistula, anorectal anomaly - high without fistula, low anorectal anomaly 
with anocutaneous fistula, low anorectal anomaly with anovestibular fistula, low anorectal anomaly 
with fistula (other specify), congenital absence atresia / stenosis anus without fistula, anus - 
imperforate, imperforate anus, low anorectal anomaly without fistula, low anorectal anoma, 
congenital anal stenosis, persistent cloaca, exomphalos (major), exomphalos (minor), exomphalos 
malrotation, exomphalos, omphalocele, closure of gastroschisis includes closure of exomphalos, 
primary repair exomphalos, repair exomphalos using prosthesis (specify type), gastroschisis, delayed 
closure gastroschisis, primary repair gastroschisis, repair gastroschisis using prosthesis (specify type), 
silo insertion for reduction of gastroschisis, delayed closure exomphalos, cutback of covered anus, 
repair of imperforate anus (with or without vaginal, cutback of low anorectal anomaly (nixon), 
oesophageal atresia - repair of anastomotic leak, primary repair of oesophageal atresia, closure of 
recurrent tracheo-oesophageal fistula, closure of tracheooesophageal fistula, closure of tracheo-
oesophageal fistula, duodenal atresia/stenosis repair, duodenal atresia/stenosis repair (von)  
Other severe congenital conditions, lethal or requiring early surgical intervention 
 Cardiac and circulatory system 
Congenital malformations of cardiac chambers and connections, common arterial trunk (truncus 
malformation), truncus arteriosus, double outlet right ventricle (dorv), double outlet left ventricle 
(dolv), dextrotransposition of aorta, transposition great arteries (tga), transposition of the great 
vessels (tga), double inlet ventricle (dilv), discordant atrioventricular connection, isomerism of atrial 
appendages, atrial isomerism & asplenia, atrial isomerism with asplenia, atrial isomerism with 
polyspenia, atrial isomerism, other congenital malforms of cardiac chambers and connections, 
congenital malforms of cardiac chambers and connections unspec, complete atrioventricular septal 

15 
 
defect, atrio-ventricular septal defect (avsd), atrioventricular septal defect (avsd), tetralogy of fallot, 
atrium single, ventricle single, congenital malformations of pulmonary and tricuspid valves, 
pulmonary valve atresia, congenital pulmonary valve stenosis, pulmonary valve stenosis (ps), 
congenital pulmonary valve insufficiency, other congenital malformations of pulmonary valve, 
congenital tricuspid atresia / stenosis, ebstein's anomaly, hypoplastic right heart syndrome, other 
congenital malformations of tricuspid valve, congenital malformation of tricuspid valve (unknown or 
unspecified cause), congenital malformations of aortic and mitral valves, congenital stenosis of 
aortic valve (as), bicuspid aortic valve, mitral atresia, congenital insufficiency of aortic valve, 
congenital mitral stenosis  (ms), hypoplastic left heart syndrome (hlh), other congenital 
malformations of aortic and mitral valves, congenital malformation of aortic and mitral valves 
unspec, coarctation of aorta, coarctation of the aorta, stenosis of aorta (as), other malformation of 
aorta, malformation of aorta, double aortic arch, hypoplasia of aortic arch, interrupted aortic arch, 
atresia of pulmonary artery, pulmonary stenosis (physiological branch stenosis), pulmonary stenosis 
- branch, other congenital malformations of great arteries, total anomalous pulmonary venous 
connection (tapvd), total anomylous pulmonary venous drainage (tapvd), blalock-taussig shunt 
Respiratory system, including diaphragmatic hernia 
Choanal atresia - bilateral, choanal atresia - unilateral (l), choanal atresia - unilateral (r), choanal 
atresia / stenosis (specify), choanal stenosis, congenital malformations of trachea and bronchus, 
congenital tracheomalacia, tracheomalacia, other congenital malformations of trachea, tracheal 
agenesis or atresia, bronchomalacia, congenital malformations of bronchus, congenital cystic lung 
(ccam), congenital cystic lung (congenital lobar emphysema), congenital cystic lung, sequestration of 
lung, congenital bronchiectasis, hypoplasia and dysplasia of lung, repair choanal atresia, congenital 
diaphragmatic hernia, congenital diaphragic hernia, morgagni diaphragmatic hernia, diaphragmatic 
hernia - left, diaphragmatic hernia - right, recurrent congenital diaphragmatic hernia, eventration of 
diaphragic hernia, eventration of the diaphragm, repair of congenital diaphragmatic hernia, 
prosthetic repair of congenital diaphragmatic hernia (specify), aplasia of the diaphragm, fetoscopic 
insertion of tracheal plug for congenital diaphragmatic hernia, other repair of diaphragmatic hernia 
(specify), other specified repair of diaphragmatic hernia, repair of diaphragmatic hernia using 
abdominal approach nec, primary repair of congenital diaphragmatic hernia, thoracoscopic repair of 
congenital diaphragmatic 
 
Brain and nervous system 
 
Does not include spina bifida occulta 
Frontal encephalocele, nasofrontal encephalocele, occipital encephalocele, encephalocoele - 
occipital, encephalocele (unknown or unspecified cause), encephalocele, meningocele (specify site), 
myelomeningocele (specify site), meningocele & hydrocephalus (specify site), thoracic spina bifida 
with hydrocephalus, lumbar spina bifida with hydrocephalus, sacral spina bifida with hydrocephalus, 
(unknown or unspecified cause) spina bifida with hydrocephalus, cervical spina bifida without 
hydrocephalus, thoracic spina bifida without hydrocephalus, lumbar spina bifida without 
hydrocephalus, sacral spina bifida without hydrocephalus, spina bifida (unknown or unspecified 
cause), spina bifida, repair of spina bifida, repair of encephalocele, anencephaly and similar 
malformations, anencephaly, craniorachischisis, iniencephaly, holoprosencephaly, closure of spinal 
myelomeningocele, closure of spinal meningocele 
 
 

16 
 
Urinary system 
Bilateral renal agenesis, renal agenesis, bilateral, potter's syndrome, autosomal recessive polycystic 
kidney - infantile, polycystic kidney, infantile type, autosomal dominant polycystic kidney in 
childhood, polycystic kidney, adult type, polycystic kidney, exstrophy of urinary bladder, bladder 
exstrophy, posterior urethral valves (puv), congenital posterior urethral valves (puv), congenital 
absence of bladder and urethra  
Other miscellaneous lethal conditions 
Thanatophoric short stature, edwards syndrome (trisomy 18), Edwards syndrome (unknown or 
unspecified cause), trisomy 18, Patau syndrome (trisomy 13), trisomy 13, sirenomelia, triploidy and 
polyploidy 
 
 
 
 

17 
 
SUPPLEMENTARY MATERIAL 2: DEFINITION OF NEC USING NNRD 
VARIABLES 
A) Severe NEC 
This method collects data from 3 tables: 
• 
Episode 
• 
Daily summary 
• 
Abdoxray 
 
Multiple fields are used to collect data about NEC diagnosis. We define Severe NEC as true if any of 
the following conditions (1-8) are met in any of the following tables: 
 
Within the Episode table: 
 
1. NEC is listed as ‘Cause of Death’ 
2. ‘Postmortem confirmation’ is true. 
3. Any of the following in ‘Gastrointestinal Diagnoses’, ‘Principle Procedures during stay’ or ‘Principal 
Diagnosis at discharge’:  
• 
LAPAROTOMY 
• 
LAPAROTOMY APPROACH NEC 
• 
COLECTOMY AND ILEOSTOMY NEC 
  
AND 
Any of the following in ‘Gastrointestinal Diagnoses’ or ‘Principal Diagnosis at discharge’: 
• 
NECROTISING ENTEROCOLITIS 
• 
NECROTIZING ENTEROCOLITIS 
• 
NECROTIZING ENTEROCOLITIS – CONFIRMED 
• 
NECROTISING ENTEROCOLITIS – CONFIRMED 
• 
NECROTISING ENTEROCOLITIS – PERFORATED 
• 
NECROTIZING ENTEROCOLITIS – PERFORATED 
• 
NECROTISING ENTEROCOLITIS - PROVEN (ON XRAY OR AT SURGERY) 
• 
NECROTIZING ENTEROCOLITIS - PROVEN (ON XRAY OR AT SURGERY 
 
4. Any of the following in ‘Gastrointestinal Diagnoses’ or ‘Principal Diagnosis at discharge’: 
• 
NECROTISING ENTEROCOLITIS 
• 
NECROTIZING ENTEROCOLITIS 
• 
NECROTIZING ENTEROCOLITIS – CONFIRMED 
• 
NECROTISING ENTEROCOLITIS – CONFIRMED 
• 
NECROTISING ENTEROCOLITIS – PERFORATED 
• 
NECROTIZING ENTEROCOLITIS – PERFORATED 
• 
NECROTISING ENTEROCOLITIS - PROVEN (ON XRAY OR AT SURGERY) 
• 
NECROTIZING ENTEROCOLITIS - PROVEN (ON XRAY OR AT SURGERY 
 
AND 
‘Discharge Destination’ listed as Death. 

18 
 
 
5. Any of the following in ‘Gastrointestinal Diagnoses’ or ‘Principal Diagnosis at discharge’: 
NECROTISING ENTEROCOLITIS – PERFORATED 
 
NECROTIZING ENTEROCOLITIS – PERFORATED 
 
Within the Daily summary table 
 
6. Surgery is listed on any day in ‘NEC Treatment’.  
AND  
Within the Episodes table 
Any of the following in ‘Gastrointestinal Diagnoses’ or ‘Principal Diagnosis at discharge’: 
• 
NECROTISING ENTEROCOLITIS 
• 
NECROTIZING ENTEROCOLITIS 
• 
NECROTIZING ENTEROCOLITIS – CONFIRMED 
• 
NECROTISING ENTEROCOLITIS – CONFIRMED 
• 
NECROTISING ENTEROCOLITIS – PERFORATED 
• 
NECROTIZING ENTEROCOLITIS – PERFORATED 
• 
NECROTISING ENTEROCOLITIS - PROVEN (ON XRAY OR AT SURGERY) 
• 
NECROTIZING ENTEROCOLITIS - PROVEN (ON XRAY OR AT SURGERY 
• 
 
Within the Abdoxray table 
7. ‘Yes’ is listed under ‘Laparotomy Performed’ and ‘Yes’ listed in ‘Histology Confirmation NEC’ 
8.  ‘Yes’ listed in ‘Visual Inspection Confirmation NEC’ 
 
B) NNAP defined NEC 
The NNAP definition of NEC, first ensures infants are born at less than 32 weeks gestation and 
survived to 48 hours using data from the Episodes table (Variables admittimeanon and  
dischtimeanon). 
 
Once the cohort of infants is established, we categorised NEC using the ‘NEC diagnosed at discharge’ 
fields from the Episodes table. 
 
/*Confirmed NEC*/ 
*NEC diagnosis confirmed by clinical signs; 
if NECDiagnosis = 1 and NECDiagBasedOn =10 and clinicalfeatures ^= .  and radiographicfeatures 
^ = . then NEC=8;   
if NECDiagnosis = 1 and NECDiagBasedOn =11 then NEC=8;* NEC confirmed by surgery; 
if NECDiagnosis = 1 and NECDiagBasedOn =12 then NEC=8; *NEC confirmed by post mortem; 
if NECDiagBasedOn=10 and clinicalfeatures ^= .  and radiographicfeatures ^ = . then NEC=8; 
*clinical signs; 
if NECDiagBasedOn in (11,12) then NEC=8; *postmortem or surgery; 

19 
 
 
/*No NEC*/ 
if NECDiagnosis =0 then do; 
if finaldischarge = 3 then NEC=7; *No NEC but died; 
if finaldischarge ^=3 then NEC=6; *No NEC but didnt die; 
Where NECdiagnosis is ticked yes but missing clinical/radiographic features confirmation this 
was treated as No NEC. 
Where NEC diagnosis is ticked yes but no further basis for that diagnosis, treated as no NEC 
if NECDiagnosis = 1 and NECDiagBasedOn = . and finaldischarge= 3 then NEC=4; /*Where infant 
died*/ 
Infants with no NEC data (NNRD fields NECDiagnosis  and NECDiagBasedOn ) entered are treated 
as 'Missing’ 
Once an episodic view of the NEC variables has been captured, it is now necessary to look across 
all the episodes of a infant for a confirmed NEC diagnosis: 
 
Maximise NEC status over all episodes for each infant. Coded as 
Missing if NEC_Diagnosis = 0 then Final_NEC=0; OR if NEC_Diagnosis = 1 then Final_NEC=1;  
NO NEC if NEC_Diagnosis in (2,3,6) then Final_NEC=2; OR if NEC_Diagnosis in (4,5,7) then 
Final_NEC=3; /*No NEC but died*/ 
NEC present if NEC_Diagnosis = 8 then Final_NEC=4 
C) Pragmatic NEC 
We define pragmatic NEC as present if either of the following conditions are met in any of the 
following tables: 
 
Within the Daily summary table: 
1. Surgery or conservative treatment is listed on any day in ‘NEC Treatment’ 
AND 
Being nil by mouth (confirmed by ‘DayEnteralFeeds’, ‘FormulaName’, ‘FeedingMethod’, 
‘VolumeMilk’) and any of the following in ‘DrugsDay’ for 5 consecutive days: 
• 
'BENZYL PENICILLIN' 
• 
'AUGMENTIN' 
• 
 'FLUCLOXICILLIN' 
• 
 'FLUCLOXACILLIN' 
• 
 'GENTAMICIN' 
• 
'CO-AMOXICLAV' 
• 
 'COAMOXICLAV' 
• 
 'CIPROFLOXACIN' 
• 
 'NETILMICIN' 

20 
 
• 
 'AMIKACIN' 
• 
 'TAZOCIN' 
• 
 'METRONIDAZOLE' 
• 
 'VANCOMYCIN' 
• 
 'CEFOTAXIME' 
• 
 'AMPICILLIN' 
• 
 'CEFUROXIME' 
• 
 'CEFTAZIDIME' 
• 
 'CEFTRIAXONE' 
• 
 'PIPERACILLIN' 
• 
 'OFLACILLIN' 
• 
 'AZLOCILLIN' 
• 
 'LINEZOLID' 
• 
 'CEFALEXIN' 
• 
 'AMOXICILLIN' 
• 
 'MEROPENEM' 
• 
 'IMEPENEM' 
• 
 'IMIPENEM' 
 
 
Within the Daily summary table: 
2. Any of the following in ‘DiagnosesDay’: 
• 
NECROTISING ENTEROCOLITIS* 
• 
NECROTIZING ENTEROCOLITIS* 
OR 
Any of the following in ‘Code’ in the Diagnosis table: 
• 
1010683 
• 
10708 
• 
15809 
AND 
Being nil by mouth (confirmed by ‘DayEnteralFeeds’, ‘FormulaName’, ‘FeedingMethod’, 
‘VolumeMilk’) and any of the following in ‘DrugsDay’ for 5 consecutive days: 
• 
'BENZYL PENICILLIN' 
• 
'AUGMENTIN' 
• 
 'FLUCLOXICILLIN' 
• 
 'FLUCLOXACILLIN' 
• 
 'GENTAMICIN' 
• 
'CO-AMOXICLAV' 
• 
 'COAMOXICLAV' 
• 
 'CIPROFLOXACIN' 
• 
 'NETILMICIN' 
• 
 'AMIKACIN' 
• 
 'TAZOCIN' 

21 
 
• 
 'METRONIDAZOLE' 
• 
 'VANCOMYCIN' 
• 
 'CEFOTAXIME' 
• 
 'AMPICILLIN' 
• 
 'CEFUROXIME' 
• 
 'CEFTAZIDIME' 
• 
 'CEFTRIAXONE' 
• 
 'PIPERACILLIN' 
• 
 'OFLACILLIN' 
• 
 'AZLOCILLIN' 
• 
 'LINEZOLID' 
• 
 'CEFALEXIN' 
• 
 'AMOXICILLIN' 
• 
 'MEROPENEM' 
• 
 'IMEPENEM' 
• 
 'IMIPENEM' 
 
 

22 
 
SUPPLEMENTARY MATERIAL 3: DEFINITIONS OF OTHER 
SECONDARY OUTCOMES 
These will be defined as: 
▪ 
Survival without severe NEC where severe NEC is defined as per the study’s primary outcome 
▪ 
Survival without severe NEC or late onset sepsis where severe NEC is defined as per the study’s 
primary outcome and late onset sepsis is defined as per the study’s secondary outcome. 
▪ 
Survival without any NEC where NEC is defined as per the study’s secondary outcome, NNAP 
defined NEC. 
▪ 
Severe brain injury (defined as either left or right grade 3 or 4 intra-ventricular haemorrhage or 
cystic periventricular leukomalacia) 
▪ 
Treated retinopathy of prematurity (defined as cryotherapy, laser therapy or injection of anti-
vascular endothelial growth factor therapy for ROP in either or both eyes). 
▪ 
Bronchopulmonary dysplasia (defined as any respiratory or ventilatory support or supplemental 
oxygen at 36 weeks postmenstrual age) 
▪ 
Severe bronchopulmonary dysplasia (defined as ventilation via endotracheal tube or 
tracheostomy, and excluding non-invasive support or CPAP, at 36 weeks postmenstrual age) 
▪ 
Time to full feeds: defined as the day of life when the infant first has three consecutive days 
without any parenteral nutrition or fluid (i.e. no parenteral nutrition or intravenous dextrose);  
 
 

23 
 
SUPPLEMENTARY MATERIAL 4: CODE LIST FOR THE DEFINITION OF 
LATE ONSET SEPSIS IN THE NNRD FIELD 
“PrincipalDiagnosesAtDischarge” 
 
sepsis - confirmed bacterial (gram positive) 
sepsis / septicaemia - confirmed with +ve microbiology 
e.coli sepsis / septicaemia 
candida sepsis / septicaemia 
group b streptococcal sepsis / septicaemia (gbs) 
staphylococcal sepsis / septicaemia 
staph. aureus sepsis / septicaemia 
sepsis / septicaemia - specified - klebsiella sp. 
sepsis / septicaemia - specified - enterobacter sp. 
sepsis / septicaemia - specified - pseudomonas sp. 
extended beta lactamase coliform infection/sepsis 
listeria sepsis / septicaemia / disseminated 
sepsis - confirmed bacterial (streptococci b positive) 
sepsis - confirmed bacterial (streptococci positive) 
streptococcal sepsis / septicaemia 
salmonella sepsis 
sepsis due to streptococcus 
umbilical sepsis / septicaemia- group b streptococcus 
 
 
 

24 
 
SUPPLEMENTARY MATERIAL 5: BUILDING THE PROPENSITY SCORE 
MODEL  
In this study we will match pairs of infants on their propensity score (a linear function of the 
covariates that were included in the propensity score model). Following the DAG, we would also 
have included neonatal unit as a highly important variable. However, probiotics are a prophylactic 
and therefore in units that use probiotics, all infants exposed to the same intervention. 
Consequently it will be very difficult to find matches who were not exposed to probiotics within the 
same unit. Importantly, infants who are not given probiotics in a probiotic centre are likely to have 
other very different characteristics e.g. be sicker, less stable or on a palliative care pathway. To 
control for some important unit level differences we choose instead to include two variables, Level 
of Care (intensive care, high dependency care, special care or normal care) and hospital network, as 
highly important variables. 
Logistic regression models will be fitted with all the critically important and highly important 
variables plus each of the moderately important variables added individually. The model with the 
largest value of the chi-squared statistic will be adopted if the test statistic exceeded 1.0. This cycle 
will be repeated, adding each remaining moderately important background variable individually, 
until none of the chi-squared test statistics exceed 1 or until all variables have been included in the 
model. We will assess the model containing the main effects for evidence of collinearity. Any 
variables where the variance inflation factors exceed five will be excluded.  
 
Interactions between background variables will also be included in the propensity score model. To 
identify the interactions for inclusion, we will sort the main effects included in the model by the 
absolute value of their t-ratios. Starting with the variable with the highest t-ratio we will examine all 
potential interactions with that variable. Continuous variables may interact with themselves, but 
binary and categorical variables may not. Potential interactions will be added individually to the 
model and the two interactions with the largest value of the chi-squared statistic, are selected if the 
test statistic exceeds 2.71 (implying significance at the 5% level for a two tailed test with 1 degree of 
freedom). This process will be repeated for each of the main effects already selected for inclusion in 
the propensity score model.