Transcript poster_for_IFmeetin

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Stool-associated microbiome of infants with surgical short bowel
•Conrad R. Cole MD, MPH, MSc 1, Charles E. Robertson PhD 2, Daniel N. Frank PhD 2, and Thomas
R. Ziegler MD 3
•1 Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA, 2University of Colorado Boulder, Boulder, Colorado
3Emory
University School of Medicine, Atlanta, Georgia, USA
Introduction
Methods
Results
•Necrotizing enterocolitis (NEC)
is a major predisposing factor for
surgical short bowel syndrome
(SBS) and intestinal failure in
infants.
• A sample size of 10 patients (6
boys and 4 girls were recruited.
• Median age of 7 months (range 2-24
months)
• Diagnosis of NEC with Hx of
intestinal resection and dependent
on PN for > 90 days
• Residual small bowel length ranged
from 25 - 60 cm, which represented
approximately 20% of expected
length of small bowel for age.
•Patients with SBS and intestinal
failure are dependent on
prolonged parenteral nutrition
(PN).
• Stool samples were collected from
these patients at baseline and
following a CLA-BSI.
• Broad-range 16S rRNA gene
PCR and pyrosequencing was
successful in all SBS subjects
and controls, with median
values of 1052 (979 - 1171)
and 1251 (1233 - 1341)
pyrosequences generated per
subject, respectively (Table 1).
•The prolonged use of PN and
minimal enteral nutrition,
although life-saving, are
associated with multiple
complications including recurrent
central line associated blood
stream infections (CLA-BSI),
small bowel bacterial overgrowth,
and increased mortality.
•CLA-BSI is a significant
contributor to the morbidity and
mortality of these children.
•Gut permeability and gut
associated bacteria may
contribute to CLA-BSI. The aim
of this study was to gain insight
into the microbial diversity of
stool of these infant and assess if
there is any relationship with
CLA-BSI.
Aim
The aim of this study was to gain
insight into the microbial diversity of
the stool of infants with intestinal failure
due to surgical resection from NEC.
We also assessed if there was any
relationship with bacteria cultured
during CLA-BSI and the stool
microbiome around the time of the
episode leading to the diagnosis of
CLA-BSI.
• A comparison group of 9 agematched healthy children without
any history of intestinal resection
were also recruited as controls and
they provided a single stool
sample.
• Stools were collected into a sterile
container and stored at -80°F until
analysis.
• The institutional review boards of
Emory University School of
Medicine, Atlanta GA and
Cincinnati Children's Hospital
Medical Center, Cincinnati, OH
approved this protocol
• Multiplexed Pyrosequencing: DNA
was extracted using a chemical and
mechanical lysis protocol
• PCR reactions were performed in
duplicate for each specimen
• Taxonomy-based operational
taxonomic units (OTUs) were
assembled by grouping sequences
Between-group differences in
biodiversity, prevalence, and
relative abundance of taxonomic
OTUs were assessed by nonparametric Kruskal-Wallis tests
using the R statistical package.
• Microbiome data
Inclusion Criteria
• Children less than 2 years of age
• History of SBS due to small bowel
resection following the diagnosis of
NEC
• Dependent on PN
• SBS was defined as dependence on
PN for at least 6 weeks with bowel
length (measured along the antimesenteric border from the ligament
of Treitz) of less than 50% of
estimated normal small bowel length
for age
Support by National Institutes of Health grant 1R21DK088027-01A1 to Conrad R. Cole
• The majority of SBS cases
(90%) had at least 1 episode of
CLA-BSI
• Organisms cultured in the
blood during CLA-BSI were
present in the stool of majority
of the subjects (Table 2).
• Klebsiella pneumonia was the
most common bacteria
cultured (35% of cases).
• Klebsiella spp. were
significantly more abundant in
the stools of SBS cases
compared with controls (19.7%
vs 2.3% relative abundance;
p = 0.014)
Table 1. Bacterial Diversity in Short Bowel Syndrome 1
Controls (N=9)
2
Taxa
Prevalence3 Abundance4
Firmicutes
100.0
51.5
Clostridium
44.4
2.7
Enterococcus
77.8
7.8
Lactobacillus
22.2
0.4
Lactococcus
22.2
1.5
Leuconostoc
0.0
0.0
Megasphaera
11.1
0.1
Peptostreptococcus
11.1
0.0
Roseburia
11.1
1.6
Staphylococcus
55.6
23.9
Streptococcus
77.8
7.9
Veillonella
44.4
3.7
Proteobacteria
77.8
29.8
Citrobacter
55.6
1.9
Dickeya
22.2
6.8
Enterobacter
66.7
5.5
Escherichia
55.6
4.6
Klebsiella
33.3
2.3
Pectobacterium
22.2
1.0
Raoultella
33.3
7.4
Bacteroidetes
33.3
10.1
Bacteroides
33.3
10.1
Actinobacteria
44.4
7.8
Bifidobacterium
22.2
7.7
Other
22.2
0.7
Sequences/Subject5
Good's Coverage6
Observed OTUs (Sobs)
Estimated OTUs (Schao1)
Shannon diversity
Shannon evenness
1251 (1233-1341)
96.8% (96.4 - 96.9)
38.4 (37.4 - 43.7)
70.2 (66.9 - 85.5)
3.1 (2.7 - 3.7)
59.0% (48.7 - 65.9)
SBS Cases (N=10)
Prevalence3 Abundance4
100.0
39.7
20.0
0.1
80.0
12.6
40.0
10.9
10.0
0.3
40.0*
1.4**
20.0
2.0
20.0
2.1
20.0
0.1
50.0
2.5
80.0
6.2
80.0
2.3
90.0
52.7
70.0
1.3
60.0
6.2
80.0
8.9
80.0
1.8
80.0*
19.7**
30.0
0.7
70.0
7.8*
40.0
4.7
10.0
4.2
70.0
2.1
40.0
0.8
40.0
0.8
1052 (979 - 1171)
95.7% (94.6 - 96.8)
48.2 (38.1 - 56.3)
90.3 (61.8 - 121.8)
3.0 (2.3 - 3.3)
53.6% (45.5 - 58.4)
1
Measured by phylogenetic distributions of 16S rRNA sequences in stool samples.
Identities of microorganisms inferred from Ribosomal Database Project Classifier. Phyla are in bold
while genera are italicized.
3
Percentage of subjects with taxa present (i.e., prevalence). Taxa that differ in prevalence between
groups are indicated (*: P < 0.1 for Fisher Exact Test).
4
Mean relative abundance of sequences classified to taxa. Taxa that differ in abundance between groups
are indicated (**: P < 0.05; *: P < 0.1 for Kruskal-Wallis Test).
5
Median sequence/subjects (Interquartile Range) analyzed in each category.
2
6
Estimates of OTU coverage, richness, and diversity were calculated using 97% OTUs. Tabulated values
are medians (Interquartile Range) obtained through 1000 boot-strap replicates.
Table 2. Concordance of blood culture and stool pyrosequencing
Subject
Cultured Organisms
Detected in Stool
1
Enterobacter spp.
+
Staphylococcus aureus
Staphyloococcus hominis
2
Staphylococcus epidermidis
+
3
Viridans streptococcus
+
Klebsiella spp.
Staphylococcus epidermidis
4
Klebsiella spp.
+
Enterococcus spp.
+
Staphylococcus epidermidis
5
Staphylococcus epidermidis
6
Enterobacter spp.
+
Enterococcus spp.
+
Klebsiella spp.
+
Leuconostoc spp.
Staphylococcus epidermidis
Staphyloococcus hominis
7
Serratia spp.
+
9
Enterococcus spp.
+
10
Enterobacter spp.
+
Enterococcus spp.
Klebsiella spp.
+
Leuconostoc spp.
Pseudomonas spp.
Staphylococcus epidermidis
11
None
n.a.