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

emccomb@som.umaryland.edu ; *bma@som.umaryland.edu

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

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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