Changes in the Gut Mycome and Microbiome during

Necrotizing Enterocolitis-like Murine Intestinal Injury.

Student:

Geerte Beesems, S1885243

Faculty supervisor:

Girbe Buist

Rijksuniversiteit Groningen

University of South Florida supervisors:

Dr. Robert Deschenes

Dr. Akhil Maheshwari

University of South Florida ǀ Morsani College of Medicine

2

Summary

INTRODUCTION – Necrotizing enterocolitis (NEC) is a gastrointestinal crisis in the newborn

infant. NEC is among the most common and devastating diseases in newborns with a mortality

up to 30% and still one of the most difficult to eradicate. NEC is therefore a priority for research.

Unfortunately, the pathophysiology of NEC is not completely understood. A multifactorial cause

is suggested in previous research, most investigative attention has remained focused on the role

of bacterial flora, and increasing evidence indicates that luminal fungi may also play a role. In

this study, the difference in the microbiome and mycobiome have been monitored using a proven

mouse model to establish the best techniques for detecting disease progression in treated versus

control mice.

MATERIAL AND METHODS – A research set up was designed and primer pairs selected. Ten

day old mouse pups were used to mimic NEC and the gut including feces was harvested for DNA

extraction. The DNA was then used for next generation sequencing in the Miseq. The DNA

sequences were analyzed using BaseSpace and QIIME.

RESULTS – The research showed some obvious differences in the microbiome between the

control versus the treated group. Where some species were only prevalent in the Control group

and other species only prevalent in the Treated group. Due to a low number of reads off the

mycobiome in the samples, no differences in the mycobiome could be detected.

CONCLUSION AND DISCUSSION – Some of the results of this research are also found in

previous research. This could indicate the pathological and/or preventive function of several

species. The mycobiome was inconclusive and more research needs to be done on this topic.

INTRODUCTIE – Necrotizerende Enterocolitis (NEC) is een gastrointestinale crisis in de

pasgeborene. NEC is een van de meest voorkomende verwoestende ziekten in pasgeborene met

een mortaliteit die oploopt tot 30% en blijkt nog steeds een van de moeilijkste ziektebeelden om

uit te roeien. Dit maakt NEC een prioriteit voor onderzoek. Helaas is de pathofysiologie van NEC

nog niet volledig begrepen. Eerder onderzoekt suggereert een multifactoriële oorzaak, het meeste

onderzoek tot nu toe heeft zich gefocust op de rol van bacteriën, maar steeds meer bewijs komt

voor de rol van schimmels en gisten. Deze studie laat de rol van het microbioom en mycobioom

zien bij NEC, gebruik makende van een muismodel.

MATERIAAL EN METHODEN – De opmaak van het onderzoek was ontworpen en primer

paren werden gekozen. Tien dagen oude muizen zijn gebruikt om de ziekte na te bootsen en de

darmen met feces werden geoogst voor DNA extractie. Het DNA is gebruikt voor next generation

sequencing met behulp van de Miseq. De hieruit gekomen sequenties zijn geanalyseerd gebruik

makende van Basespace en QIIME.

RESULTATEN – Dit onderzoek liet een aantal hele duidelijke verschillen in het microbioom

zien tussen de behandelde groep tegenover de controle groep. Sommige soorten werden alleen

aangetoond in de behandelde groep en sommige soorten alleen in de controle groep. Door een

laag aantal reads van het mycobioom konden hier geen verschillen worden aangetoond.

CONCLUSIE EN DISCUSSIE – Sommige van deze resultaten zijn in overeenkomst met eerder

onderzoek en zou een pathologische en/of beschermende functie van verschillende soorten

kunnen laten zien. Over het mycobioom kon niks worden gezegd en meer onderzoek is nodig op

dit gebied.

3

Table of contents

Introduction 4

Materials and Methods 6

Research set up and preliminary research 6

Murine model, sample processing and DNA isolation 8

PCR amplification, Purification, quantification and sequencing 9

Data Analysis 9

Results 10

DNA isolation 10

Primer selection and PCR 10

Murine Model 11

Micriobiome 12

Mycobiome 17

Conclusion and Discussion 19

References 20

Supplementary table 23

4

Introduction

Necrotizing enterocolitis (NEC) is a gastrointestinal crisis in the newborn infant. It is a disorder

characterized by various signs and symptoms including, but not limited to: ischemic necrosis of

the intestinal mucosa, inflammation, feeding intolerance, abdominal distension, bloody stools

after 8 to 10 days of age and the potential for perforation of the bowel with septic shock.

Although early recognition and treatment of this disorder have improved the clinical outcome,

NEC is among the most common and devastating diseases in newborns with a mortality up to

30% and still one of the most difficult to eradicate. NEC is therefore a priority for research1-2

.

NEC is the most common gastrointestinal emergency in neonate intensive care units. In the

United States, the incidence ranges between 1 and 7.7% of all the admissions. The incidence of

NEC decreases with increasing gestational age and increasing birth weight. The mortality rates

are also inversely related with birth weight and gestational age3-5

. Even though the majority of

infants with NEC are preterm infants with a low birth weight, approximately 13% of cases occur

in term infants. However, the term infants who develop NEC typically have an underlying

preexisting illness2,6

.

It is common in all very-low-birth-weight infants to have intermittent gastrointestinal symptoms.

These include “concern causing symptoms” such as bloody stool, feeding intolerance and

abdominal distension, however most do not have NEC. Once NEC is definitive, medical

intervention is required and often based on the clinical presentation of the disease. This includes

abdominal decompression, antibiotics, laparotomy and peritoneal drainage. Once surgical

intervention is necessary, however, increases in mortality and morbidity dominate the outcomes.

This is a finding that highlights the need for effective prevention of NEC7-9

.

As soon as NEC is suspected, medical management is initiated. The medical management

consists of antibiotic therapy, supportive care and close monitoring of the infant. The supportive

care and antibiotic therapy focus on limiting the progression and preventing the disease to arise

and is initiated in all patients. Radiologic and laboratory studies monitor the course of the disease

and are used to determine the clinical improvement or the deterioration of the patient with the

disease. Progression of the disease in patients undergoing antibiotic therapy also serves as an

indication for the requirement of surgical intervention10-11

.

Unfortunately, the pathophysiology of NEC is not completely understood. However, there are

strong epidemiologic observations which suggest a multifactorial cause. The combination of

intestinal immaturity, an imbalance of the microvascular tone and the likelihood of abnormal

microbial colonization in the intestine are all factors that have the potential for progression of the

disease. Existing information emphasizes a central pathophysiological role of gut microbial flora

in NEC, driving both tissue damage as well as inflammatory injury. However, specific pathogens

have not been causally-associated with NEC. Although most investigative attention has remained

focused on the role of bacterial flora, increasing evidence indicates that luminal fungi may also

play a role1,12

.

5

Previous animal research came up with several animal models for NEC-like symptoms. Rabbits,

rats, C57BL/6 mice and piglets were used. The articles concluded that these animals all can be

used as a model for NEC, however none of the articles looked into the microbiome and

mycobiome of the disease. They all focused on the histological changes that occur with NEC13-16

.

Previous research in preterm infants showed a beneficial role of probiotics and breastfeeding in

the prevention of NEC, also NEC does not occur in germ free mice which can be a lead to the

differences in the microbiome and mycobiome of the sick versus the healthy preterm17-19,33

.

Several small studies looked into the differences in microbiome in preterm infants with NEC and

implied several differences in the microbiome of NEC versus control20

. However, the specific

findings differ significantly among those studies. One study showed an increase of

Proteobacteria and a decrease in Firmicutes in NEC cases collected one week and <72 h prior to

NEC diagnosis34

. Another study demonstrated a tendency toward a lower alpha-proteobacterial

diversity in infants, who later developed NEC35

. The lower bacterial diversity of NEC cases

versus controls was confirmed recently. Microbial diversity and Clostridia abundance and

prevalence even decreased with increasing severity of NEC36

.

In this study, the difference in the microbiome and mycobiome have been monitored using a

proven mouse model to establish the best techniques for detecting disease progression in treated

versus control mice21

. The treated mice were given TNBS to induce NEC-like symptoms and

sacrificed for harvesting the gut.

6

Materials and methods

The studies were approved by the Institutional Animal Care and Use Committee

(https://www.aalas.org/iacuc), which provides regulatory oversight over all animal

experimentation.

Research set up and preliminary research

To answer the research question, a global research set up had to be created (Figure 1). An

existing mouse model using trinitrobenzene sulfonic acid (TNBS) for the mimicking of the

disease was used (n=24)21

. It was decided to include the ileum and the proximal colon of the

mouse pups. This way differences between the ileum and colon within a mouse and between the

mice could be determined. The DNA of these parts of the bowel including the feces was extracted

and used for further sequencing.

7

Figure 1. Flowchart of the research set up.

DNA isolation

The mouse pup gut has a really low weight (<10µg) and therefore a DNA isolation method had to

be selected that resulted in the isolation of a minimal amount of approximately 100 ng/μl

bacterial and fungal DNA at the same time. The average size of the DNA fragments had to be

around 300bp to be able to determine the nucleotide sequence using the Miseq. Qiagen Gentra

Puregene Yeast/Bact. Kit, QIAamp Fast DNA Stool Mini Kit, ZR fungal/bacterial DNA

microprep kit were tested according to manufacturers’ protocol. Thereafter the isolated DNA was

analyzed after electrophoresis in an 1% agarose gel. Hereafter the samples with the right sizes

DNA fragments were used in a PCR with bacterial and a PCR with fungal primers. In this way all

the different kits and methods were checked.

Primer testing and selection

For the amplification of the bacterial 16S sequnces the universal primer pair 341F (5′-

CCTAYGGGRBGCASCAG) and 806R (5′-GGACTACNNGGGTATCTAAT) was used

following the manufacturer’s protocol, (Illumina Inc, USA). Currently there is no existing

universal primer pair for the amplification of fungal 18S regions. In different articles primer pairs

have been used for PCR of either the ITS2 and 18S regions. Based on these articles the expected

fragment length and the region of the described pairs, 7 primer pairs were selected to be tested for

the fungal regions23-25

. Important were the fragment lengths and the amount of DNA after PCR

for later use in the Miseq. After several test PCRs the best PCR conditions were determined (see PCR amplification, Purification, quantification and sequencing) the best primer pair was; NS1

(5’-GTAGTCATATGCTTGTCTC) and SR3 (5’-GAAAGTTGATAGGGCT) and used for

processing on the Illumina Miseq (Figure 2).

8

Figure 2. Work schedule for the primer pair selection.

Murine model, sample processing and DNA isolation

Tissue samples were obtained from 10 day old C57/BL6 mouse pups (n=27 animals). NEC-like

gut mucosal injury was induced in mouse pups (n=14 animals) by administering the haptenic

agent TNBS by gavage and enema on day 10. Animals were sacrificed 24 hours after receiving

TNBS and the proximal 2 cm of the ascending colon and the distal ileum were harvested for

high-throughput studies to characterize the mycobiome and microbiome in control and TNBS-

treated animals. During harvesting the level of injury was visually inspected and defined with a

platelet count since NEC is correlated with a decrease in platelet count22

. Treated animals without

tissue damage were excluded for further research (n=3).

9

Genomic DNA from the different samples was extracted using the ZR fungal/bacterial DNA

microprep kit (Zymo Research, USA) following manufacturer’s instructions. DNA concentration

was determined using NanoDrop (Thermo Scientific, Wilmington, USA).

PCR amplification, Purification, quantification and sequencing

The universal primer set 341F (5′-CCTAYGGGRBGCASCAG) and 806R (5′-

GGACTACNNGGGTATCTAAT) was used for amplification of the V3–V4 regions of 16S

rDNA. For the V-regions of the 18S DNA the primer set NS1 (5’-

GTAGTCATATGCTTGTCTC) and SR3 (5’-GAAAGTTGATAGGGCT) was used. The

amplicon PCR was carried out in a 25µl reaction volume with 12.5µl 2x KAPA HiFi HotStart

ReadyMix (KAPA Biosystems, UK), 5µl 1μM forward and reverse primers, and 12.5 ng template

DNA. For the microbial PCR thermal cycling manufacturers’ protocol was used. For the

mycobial PCR The thermal cycling consisted of denaturation at 95 °C for 3 min, followed by 25

cycles of 95 °C for 30s, 55 °C for 30s, and 65 °C for 30s, and finally 72 °C for 5 min. PCR

products were analyzed in an 1% agarose gel. The PCR products were cleaned using AMPure XP

beads (Agencourt Bioscience Corporation, Massachusetts USA) to purify the DNA amplicons

from DNA fragments shorter than 100bp. Sequencing libraries were labeled with two indeces

creating 96 different barcodes using Nextera DNA Library Preparation Kit following

manufacturer’s protocol (Illumina Inc., USA). The quality of the prepared DNA was assessed

with a Qubit Fluorometer (Thermo Scientific, USA) and the fragment length was determined by

visual inspection of the gel. The libraries were sequenced on an Illumina Miseq platform.

Data analysis

The analysis of the microbiome sequences was done using Basespace (illumine Inc., USA). For

the mycobiome data, the FastQ files were retrieved and the primer sequences removed from the

start of pair-end sequences and joined using Pndaseq. All the sequences that had zero base pairs

from the joined reads were removed using a Perl script. All reads with a basepair length over 100

bp were used for further analysis. The reference data from Silva 104 with sequences clustered at

97% identity were used to generate the mycobiome.

10

Results

DNA isolation

To determine which isolation procedure should be used to isolate bacterial and fungal DNA

simultaneously the Qiagen Gentra Puregene Yeast/Bact Kit, QIAamp Fast DNA Stool Mini Kit,

ZR fungal/bacterial DNA microprep kit and the regular DNA extraction method for bacteria with

an extra heating step were tested and compared. After gel electrophoresis it turned out that the

Qiagen Gentra Puregene Yeast/Bact kit did not produce enough DNA when using the small

sample size, this was shown in a low quantification of the concentration using the Nanodrop

(Thermo Fisher Scientific Inc, USA). The QIAmp Fast DNA Stool Mini did not extract the

fungal DNA as the DNA was not amplified during the PCR.This could be due to the lack of a

heating step within the protocol to open the fungal cell wall. Using the ZR fungal/bacterial DNA

microprep kit resulted in extraction of bacterial and fungal DNA in decent amounts and therefore

was chosen for further use.

Primer selection and PCR As no standard primers were available for PCR of the fungal DNA primers were selected.

Finding the right primer pair for the fungal DNA was challenging. After PCR the DNA fragment

sizes had to be between 300-500bp to be able to be use the sample in the Miseq. Many of the

PCR products did not show a clear product in the agarose gel or showed a broad band instead of a

clear line. After several PCR reactions and analyses in gels, the best primer pair and PCR

conditions were selected (see methods section). The PCR conditions that were changed during

determination were number of cycles and annealing temperature. The primer pair NS1 and SR3

showed the best results (figure 3).

Figure 3. Analysis of the PCR products by gel electrophoresis after using the primer pair

NS1/SR3. Shows a PCR product of ±400bp in a clear line on the controls as well as the samples.

Number 1: 100bp ladder; Number 2: positive control on pure sample; Number 3: negative

control; Number 4: mixed sample; Number 5: mixed sample.

11

Murine model

To make sure the mouse model worked, all the samples were visually inspected on injury and two

platelet counts (mean 37.63, +/- SD 15.13) were performed per sample (table 1). The visual

inspection was included to make sure the injury was as expected with the treatment, because the

lower platelet count can be inconclusive. No injury means no visible injury, with moderate injury

blistering and eruptions in the gut were visible. Severe injury included blistering, perforations and

blood throughout the gut.

* samples were not sequenced for the microbiome.

** high platelet value most likely due to a bloodcloth.

*** Samples not included for further analysis.

Table 1. Overview of results obtained for extra check of the mouse model.

Control sample 2 showed severe injuries in the gut and the platelet count was really low,

therefore this sample was excluded from further analysis. Samples TNBS9, TNBS10 and

TNBS11 did not show any injury, therefore these samples were excluded for further analysis as

Sample ID Treatment Visual injury? Platelet count

1* Control None 27-30

2* Control Severe *** 9-10

3* Control None 43-58

4* Control None 6-9

5* Control None 42-37

6* Control None 38-38

7 Control None 7-18

8 Control None 49-36

9 Control None 31-254**

10 Control None 36-38

1 TNBS Severe 24-25

2 TNBS Moderate 28-31

3 TNBS Moderate 17-17

4 TNBS Moderate 24-25

5 TNBS Moderate 36-37

6 TNBS Moderate 24-20

7 TNBS Severe 25-26

8 TNBS Moderate 34-40

9 TNBS None*** 39-38

10 TNBS None*** 51-59

11 TNBS None*** 50-46

12 TNBS Moderate 36-30

13 TNBS Moderate 67-60

14 TNBS Moderate 49-49

12

well. The platelet count showed a significant lower number in the TNBS-treated animals

(unpaired T-test: 0,14).

Microbiome

Previous research showed a lower bacterial diversity of NEC cases versus controls, in our study

the average number of identified species in the control animal was 48,25. The average number for

the TNBS treated animals was 45,16. This shows a significant lower number of identified species

in the TNBS treated animals (unpaired T-test: 0,32).

During the sequencing PCRs it turned out that three samples did not contain enough or any DNA

to be used further. These samples were derived from the TNBS treated animals and were part of

the ileum. All other samples showed a significant higher number of species identified in the colon

versus the ileum, this was true for the TNBS treated animals, the Control animals and in general

(unpaired T-test respectively, 0.08 and 0.19). The differences in microbiome between the two

groups are visualized on the next two pages (fig 5 and fig 6). For specific numbers see

supplementary table 1.

13

Fig 5 . Overview of the microbiome of the TNBS treated animal group.

14

Fig 6 . Overview of the microbiome of the Control animal group.

15

Phylum-level

The phylum level of the results already showed a big difference between the two groups.

In both groups Firmicutes has the highest prevalence while Bacteroidetes was almost non

prevalent in the Control animals. The phylum Tenericutes was present in the TNBS treated

animals but not in the Control animals.

Class-level

Both groups show the highest prevalence of Bacilli. Bacteroidia and Spingobacteria are highly

present in TNBS treated animals, but not in the Control animals. Clostridia on the other hand was

significantly higher present in the Control animals.

Order-level

On order level Lactobacillales was the most prevalent order in both groups, however it was much

more present in the Control animals group. Bacillales, Bacteroidales, Sphingobacteriales and

Coriobacteriales were present in the TNBS-treated group and (almost) not prevalent in the

Control group. The order Clostridiales was significantly higher prevalent in the Control group.

Family-level

On family level the Lactobacillaceae were the most prevalent in both groups, however it makes

for a significant higher percentage in the Control group (43% versus 95%). The TNBS-treated

group shows a higher prevalence of the Staphylococcaceae, Enterococcaceae,

Porphyromonadaceae and Bacteroidaceae. The Control group shows a significantly higher

prevalence of the Streptococcaceae and the Lachnospiraceae.

Genus-level

On genus level Lactobacillus was the most prevalent genus in both groups, however more

prevalent in the Control group (42% versus 95%). Next to the Lactobacillus, the Pediococcus,

Streptococcus, Blautia and Escheria were significantly more prevalent in the Control group. In

contrast, the Staphylococcus, Enterococcus, Parabacteroides, Olivibacter, Bacteroides,

Pedobacter, Adlercreutzia and Dysgonomonas were significantly more prevalent in the TNBS

treated group.

Species-level

On species level Lactobacillus johnsonii was the most prevalent in both sample groups, however,

the most prevalent in the Control group (39% versus 78%). Just like the Lactobacillus vaginalis,

Lactobacillus antri, Streptococcus bovis and the Enterococcus casseliflavus. Significantly higher

prevalent in the TNBS-treated group are the Staphylococcus xylosus, Enterococcus faecalis,

Staphylococcus gallinasum, Parabacteroides goldsteinii and the Enterococcus dispar.

For some of the species the difference between the two groups was astonishing, were the

abundance in one group was high and in the other group not or very little, this is displayed in

figure 7.

16

Figure 7. Differences in 16S abundance

in the Control vs. the TNBS-treated group.

The Y-axis shows the 16S abundance per sample.

Two parts of the bowel were harvested for analysis, these were the ileum and the proximal colon.

There were significant differences between the two parts (Fig. 8). In the colon there were several

species specific detected in the Control animals; Enterococcus gallinarum, Blautia

wexlerae,Enterococcus casseliflavus, Enterococcus gallinarum and Enterococcus gilvus.

Pedobacter kwangyangensis and Parabacteroides goldsteinii were only present in the TNBS

treated group. In the ileum there were no species differences found between the two groups.

17

a ;Enterococcus gallinarum

b :Blautia wexlerae

c :Enterococcus casseliflavus

d :Enterococcus gallinarum

e :Enterococcus gilvus

f :Pedobacter kwangyangensis

g:Parabacteroides goldsteinii

Figure 8. Differences in species abundance in the colon.

Mycobiome

Less than one percent of all the reads from all the samples were reads of the 18S abundance from

fungi. This either means that there were not many fungi present in the samples, or something

went wrong. Therefore the analysis was not as detailed performed for the microbiome. Figure 9

shows the mycobiome of the mouse pups. Due to the low number of reads the reads did not show

a significant difference between the TNBS treated group versus the Control group. Also there

were no differences found between the ileum and the colon.

18

Figure 9 . Overview of the mycobiome of both groups.

19

Conclusion and Discussion

All previous research that has been done used feces only. Since this research included parts of the

bowel, the mucosa has also been used. It is known that there is a microbiome in the mucosa of the

gut, this could be a reason for the differences in our findings in comparison to previous research.

Microbiome

There are really obvious differences between the microbiomes of the control group and the TNBS

treated group, also there is a clear difference between the colonization of the colon and the

colonization of the ileum. This implies a specific role for bacteria in NEC.

The colon showed a more diverse microbiome and showed a higher number of bacteria in

comparison to the ileum. Next to that, the colon of the control group had a bigger and more

diverse microbiome compared to the TNBS treated group. This higher number of bacteria can be

explained by the pH-level in the gut. The pH in the ileum is lower than in the colon, therefore the

environment of the colon is better suited for bacteria to grow26

. The higher diversity and higher

number of bacteria in the control group shows the importance of the microbiome. As described

before, NEC is related with lower number of bacteria and a lower diversity1.

The fact that the Enterococcus faecalis was present only in the TNBS treated group, could imply

a pathological role of this bacterium. Opposite; the Lactobacillus antri, Lactobacillus vaginalis

and the Enterococcus gallinarum were only present in the Control group and therefore could

have a preventive function in the emerging of a disease.

The Lactobacilli were more present in the Control group. It is known that mouse pups under

stress do not drink as much milk as healthy non stressed mice. Therefore the colonization of the

Lactobacillus can be induced with the transfer of the microbiome of the mother’s milk when

lactating. However it is unsure if the colonization by this bacterium is caused as a result of not

eating by the mice because it is feeling sick, or as a cause of the mouse getting sick. Earlier

research did show an advantage of mother’s milk in the prevention of NEC27-29

.

Compared to human newborns, the mice from both groups, but mainly in the TNBS-treated

group, showed a really low count of gram-negative bacteria. Since it is expected that all bacteria

influence each other in the newborn gut, in order to mimic the human situation in preterm

infants, it is necessary to add these bacteria in a new mouse model. This means the mouse model

now is not a good representation of the human newborn situation and needs to be revised30-31

.

Previous research showed showed an increase of Proteobacteria and a decrease in Firmicutes in

NEC34

. Our study did show the decrease in Firmicutes, however it did not show the higher

prevalence of Proteobacteria. Another study demonstrated a tendency toward a lower alpha-

proteobacterial diversity in infants, who later developed NEC35

. We did not find this change. The

lower bacterial diversity of NEC cases versus controls was confirmed recently, also this research

shows this. Microbial diversity and Clostridia abundance and prevalence even decreased with

20

increasing severity of NEC36

. This is in agreement with our findings. So there are a few

similarities and a few differences when this research is compared to previous studies.

Mycobiome

Because of the low number of reads of the 18S region, no significant differences in the

mycobiome between the two groups could be determined. There could be multiple reasons why

there were limited reads of the 18S region. The mice are only 10 days old when sacrificed, maybe

there hasn’t been any colonization by the fungi yet. Possibly it is a problem with the primerpair.

Also for fungal DNA detection on the Miseq a higher concentration of input DNA could be

useful. So far not much is known about the mycobiome. The previous research was on humans

and not on mice and there was no literature on the mycobiome of the healthy human gut32

.

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Supplementary table 1. Exact reads and percentages of the microbiome.