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W01_0056_VW01_0055_WPMOS01_0054_WYV01_0052_R01_0049_ZMW01_0044_YQO01_0042_JTE01
_0041_FTL01_0036_ age
(weeks)1.00000.04000.0016undanceAbmium_longuBifidobacterium_longumBifidobactermium_longuBi
fidobacterium_longumBifidobactermium_longuBifidobacterveium_breBifidobactermium_longuBifidoba
cterum_longumriBifidobacteium_longumBifidobacterveium_breBifidobactereevum_brriBifidobacteveiu
m_breBifidobacterium_longumBifidobacterium_longumBifidobacterium_longumBifidobacterveum_bre
riBifidobacteium_longumBifidobacterium_longumBifidobacterium_longumBifidobacterveium_breBifido
bacterveium_breBifidobacterviium_scardoriBifidobacteveium_breBifidobacterveium_breBifidobacteru
m_longumriBifidobacteveum_breriBifidobactemum_longuriBifidobacteium_longumBifidobacterveium_
breBifidobacterveium_breBifidobacterviiium_scardoBifidobacterveium_breBifidobacterium_longumBifi
dobacterviiium_scardoBifidobactermium_longuBifidobacterium_longumBifidobacterium_longumBifido
bactermium_longuBifidobacterium_longumBifidobacterveium_breBifidobactermium_longuBifidobacter
veium_breBifidobactermum_longuriBifidobactemium_longuBifidobacterveium_breBifidobactermium_l
onguBifidobacterveium_breBifidobacterveium_breBifidobacterium_longumBifidobactermium_longuBifi
dobacterveium_breBifidobacterium_longumBifidobacterum_longumriBifidobacteium_longumBifidobac
terveium_breBifidobacterviium_scardoriBifidobactemium_longuBifidobacterium_longumBifidobacterve
ium_breBifidobacterium_longumBifidobactermum_longuriBifidobacte
Non-Cognitive Predictors of Student Success:
A Predictive Validity Comparison Between Domestic and International Students
INTRO
• “Leaky gut” (high intestinal permeability) is the proximate cause to
necrotizing enterocolitis in preterm neonates
• Intestinal barrier maturation is associated with exclusive
breastfeeding, less antibiotic exposure, and altered composition of
the gut microbiota
• Study objective: to define microbial biomarkers for improved
intestinal barrier maturation with high taxonomic resolution to
develop novel diagnostic and therapeutic strategies
STUDY DESIGN AND METHODS
MAJOR RESULTS
High-resolution evaluation of
gut microbiota associated with
intestinal maturation in early
preterm neonatesElias McComb, Mike Humphrys, Shilpa Narina, Luke
J. Tallon, Lisa Sadzewicz, Sripriya Sundararajan,
Rose Viscardi, Jacques Ravel, Bing Ma*University of Maryland Baltimore · Institute for Genome Sciences
[email protected] ; *[email protected]
Take a picture for PacBio
webinar on Sequel II
capabilities presenting our
data!
0w 1w 2w 3w
24-33w gestation preterm
neonates enrollment*
Intestinal permeability
measurement**
Daily stool in DNA/RNA
shield buffer ***
Daily dry stool ***
16S rRNA V3-V4 amplicon
Illumina sequencing
16S rRNA full-length
Pacbio Sequel II sequencing
Metagenome WGS
Pacbio Sequel II sequencing
**Intestinal permeability was measured using urine non-metabolized sugar probes lactulose and rhamnose during the first 7-10 days of life
***Stool specimen was collected daily at every stooling event
*Geographical, clinical, and nutritional information was collected for each enrolled preterm neonate
Enrollment goal: 200
Currently enrolled: 87
• UMB Accelerated Translational Incubator Pilot Grant
• PacBio 2018 SMRT grant award
Fig. 3. Relative abundance of specific bacterial taxa associated with intestinal permeability
• Bifidobacteriales and Clostridiales are most significantly associated with improved intestinal permeability.
Fig. 1: Intestinal permeability as a function of microbial community composition
• At birth, majority preterm have highly leaky gut (La/Rh > 0.05)
• At postnatal day 8, intestinal barrier matured while 21.4% preterm neonates maintained
elevated intestinal permeability (IP)
• IP correlated with increased community diversity and microbiota composition structure (Ma
et al. 2018)
* Postmenstrual age (PMA) is calculated as gestational age at birth plus postnatal age as defined previously (Grier et al., 2017).
Fig. 4: ANI clustering of full-length 16S rRNA gene sequences aggregates ASVs to generate sub-
species types
• At least 19 subspecies of B. longum and 5 B. breve were observed
• By comparing to 131 full-length 16S rRNA gene sequences of B. longum, 3 of the top most
abundant B. longum (ASV2, 29, 66) belong to B. longum subspecies infantis and B. longum
subspecies longum. These two subspecies have been previously identified to be the
“champion colonizer” of the infant gut and adapted to metabolize human milk
oligosaccharides and display anti-inflammatory activities
• Within Clostridiales and Lactobacillales, we observed 39 subspecies from Clostridiales
(clusters IV , XIVa and I) in species of Blautia faecalis, Roseburia intestinalis, Eubacterium
rectale, Ruminococcus torques, etc., and 4 subspecies of Lactobacillus fermentum, 2
subspecies of Lactobacillus salivarius, and 4 subspecies of Lactobacillus rhamnosus
Fig. 5: Phylogenetic tree of Bifidobacterium
subspecies in stool microbiota of cohort
• Multiple subspecies of Bifidobacterium can
coexist in an infant
• A single subspecies of Bifidobacterium can
be shared between several infants and that
at multiple time points
• The presence of a multiple subspecies with
an infant suggests strong environmental
adaptive advantages and high microbial
community dynamics
ONGOING• Metagenome sequencing using long-reads PacBio Sequel II sequencing generates
metagenome-assembled genomes in closed or draft forms
• Characterize genome content by sequencing metagenomes to understand strain-specific
carbohydrate metabolism potentials of Bifidobacterium and Clostridiales
• Understand the genetic basis of cross-feeding interactions between Bifidobacterium
(bifidogenic) and Clostridiales (butyrogenic)
Fig. 6: Closed, complete or nearly complete metagenome-assembled genomes were
generated using sequencing coverage between 7X-115X
Take a picture for our
first publication on this
topic
Poster #7079
• A total of 1,360 ASVs were identified
with an average sequence length of
1,462+/-14bp
• The majority of ASVs (87.6%) were
assigned to 180 species, no available
reference available for remaining ASVs
• 7,066 +/- 536 sequence/sample,
17.4+/-13.8 ASVs/sample and 5.3+/-3
species/sample were obtained
Fig. 2: ZymoBIOMICS controls sequencing on PacBio Sequel II
• Despite high abundance of Escherichia coli and Klebsiella
pneumoniae in the gut microbiota, preterm neonates with
Clostridiales and Bifidobacterium, (B. breve, B. longum subsp.
infantis, B. longum subsp. longum) have low intestinal
permeability (IP) at postnatal days 7-10.
High IP (Leaky gut)
25-28w (E
arly)
Bacillales
Bactero
idales
Bifid
obacteriales
Clostrid
iales
Entero
bacteriales
Lacto
bacillales
Propionibacteriales
Pseu
domonadales
0
3
6
9
12
0
3
6
9
12
0
3
6
9
12
Ab
un
da
nc
e
Bacillales
Bactero
idales
Bifid
obacteriales
Clostrid
iales
Entero
bacteriales
Lacto
bacillales
Propionibacteriales
Pseu
domonadales
Low IP (non-leaky gut)
Po
stmen
strual ag
e (weeks) *
29-32w (M
ediu
m)
33-35w (L
ate)
**
**
***
• PacBio Sequel II closed metagenome-assembled genomes of B.
breve reveal a battery of carbohydrate metabolism capabilities
including “bifid shunt”, indicating a capacity to consume and
convert human milk oligosaccharides (HMO) to short chain fatty
acids (SCFAs) with the potential to influence host physiology.
B. Breve JCM1192
B. Breve MAG (Subject 55, day 7)
B. Breve MAG (Subject 55, day 11)
Sample Total Bp #contig MinLen MedianLen MeanLen MaxLen N50 N50_len N90 N90_len
55-11.1 2392065 4 82560 144264 598016 1241394 1 1241394 2 923847
55-7.1 2350410 1 2350410 2350410 2350410 2350410 1 2350410 1 2350410
Sample Total Bp #sequences Mean Median Min Max N50
55-11.1 90,371,177 18,057 16,398 15,749 589 54,126 19,411
55-7.1 277,881,904 55,500 15,186 14,903 304 47,560 17,660
Table 1. Statistics of Sequel II sequence reads
Table 2. Statistics of assembly *assembly was performed using Canu-1.8, alignment to reference B.
breve genome JCM1192 was performed using MAUVE aligner. The statistics for the contigs aligned to
reference is shown below.
Preliminary analysis of the genome content of B. breve revealed a variety of oligosaccharide
transport proteins, glycosyl hydrolases genes, genes involved in the production of fucose and sialic
acid, and the unique sugar metabolic pathway “bifid shunt”. These results indicate B. breve
remarkable capacity to digest and consume human milk oligosaccharide (HMO).
FUNDING
Check out Booth #861 for
Sequencing the ZymoBIOMICS Microbial
Community Standard, we observed unbiased and
species-level taxonomic resolution
Enroll preterm infants with
24-33w gestationMeasure gut leakiness at
postnatal days 7-10
2/3. SMRTbell
library construction
Sequel II Platform
2. 27F/1492R full-length 16S rRNA PCR
Collect daily stool and neonatal
metadata postnatal days 0-21
Qiagen PowerViral DNA/RNA
adapted for Hamilton Robotics
1. 319F/806R V3V4 16S rRNA PCR
3. Sample preparation for metagenomics
1. Illumina library
construction
MiSeq Platform
(Urine non-metabolized sugar probes
lactulose and rhamnose) DNA extraction
0%
25%
50%
75%
100%
Run 1 (n=1990)
Run 2 (n=3822)
Expected
Bacillus_subtilisEnterococcus_faecalisEscherichia_coliLactobacillus_fermentumListeria_monocytogenesPseudomonas_aeruginosaSalmonella_entericaStaphylococcus_aureus
ASV_1020
ASV_1021
ASV_1042
ASV_1045
ASV_1050
ASV_1052
ASV_1084
ASV_1090
ASV_1091
ASV_1099
ASV_11
ASV_1100
ASV_1103
ASV_1135ASV_1141
ASV_1147
ASV_1156
ASV_1168
ASV_1169
ASV_1174
ASV_1189
ASV_1191
ASV_1196
ASV_1200
ASV_1206
ASV_1207
ASV_1229
ASV_1239
ASV_1248
ASV_1254
ASV_1276
ASV_1283
ASV_1285
ASV_1293
ASV_1294
ASV_1305
ASV_1310
ASV_1322ASV_1332
ASV_1334
ASV_1368
ASV_1373
ASV_2ASV_29
ASV_5
ASV_6
ASV_647
ASV_66
ASV_761
ASV_8
ASV_83
ASV_830
ASV_859
ASV_898
ASV_931
ASV_939
ASV_950ASV_956
ASV_957
ASV_974
ASV_995
Abundance
0.0016
0.0400
1.0000
Postmenstrual Age (wks)
29_32
33_35
Subject
a
a
a
a
a
a
a
a
a
01_0036_TQV
01_0041_FTL
01_0042_JTE
01_0044_YQO
01_0049_ZMW
01_0052_RYV
01_0054_WOS
01_0055_WPM
01_0056_VWW