Next-generation Toolbox for Probiotic Analysis · Next-generation Toolbox for Probiotic Analysis...

Next-generation Toolbox for

Probiotic Analysis

Christopher A. Elkins, Ph.D. Director

Division of Molecular Biology

OARSA

Wiley

Building

CFSAN HQ

Probiotic Products as Dietary

Supplements What is a dietary supplement?

“a product (other than tobacco) intended to

supplement the diet that bears or contains

one or more…dietary ingredients”

-Federal Food Drug and Cosmetic Act (FDCA) §201(ff)(1)

Dietary Ingredients • Vitamin

• Mineral

• Amino acid

• Herb or other botanical

• Dietary substance for human use to supplement the diet by increasing the total dietary intake (probiotics)

• Concentrate, metabolite, constituent, extract, or combination of any ingredient above

(FDCA §201(ff)(1))

The review of a microbial NDI focuses on:

- Identity of the microorganism

- History of use*

- Fermentation and processing

- Safety of the microorganism

• Pathogenicity

• Toxins

• Antibiotic resistance genes

- Toxicological and clinical data

New Dietary Ingredient (NDI) Review Process

Consumer’s Guides

Custom Probiotics Inc.

Label

Strain information Dose- viability

Disclaimer Drug/Disease claim

GOALS:

• To provide a better understanding of the contents of

foods that contain live microbials, the accuracy of

their label and ability to identify potential

contaminants.

• Creation of a genotypic and phenotypic database of

microbes commonly found in dietary supplements to

support whole genome sequencing, through

metagenomics, as a post-market surveillance tool.

Product

culture-independent

metagenomics culture-dependent

single colony isolates

sequence sequence

Phenotypic

characterization

GENIII

K-mer analysis

Cosmos ID

MetaPhlAn

Identification

Taxonomy

Phylogeny

K-mer analysis

Identification

Database Identification

Product

culture-dependent

single colony isolates

sequence

Phenotypic

characterization

GENIII

Taxonomy

Phylogeny

K-mer analysis

Identification

Database Identification

NDI

Arthrobacter agilis Bacteroides lipolyticum

Arthrobacter citreus Bacteriodes succinogenes

Arthrobacter globiformis Brevibacterium lipolyticum

Arthrobacter luteus Brevibacterium stationis

Arthrobacter simplex Kurthia zopfii

Acinetobacter calcoaceticus Myrothecium verrucaria

Azotobacter chroococcum Pseudomonas calcis

Azotobacter paspali Pseudomonas dentrificans

Azospirillum brasiliense Pseudomonas fluorescens

Azospirillum lipoferum Pseudomonas glathei

Bacillus brevis Phanerochaete chrysosporium

Bacillus marcerans Streptomyces fradiae

Bacillus pumilis Streptomyces cellulosae

Bacillus polymyxa Streptomyces griseoflavus

Bacillus subtilis

Lactobacillus rhamnosus

Lactobacillus acidophilus

Lactobacillus reuteri HA-188

Bifidobacterium lactis HN019

Bifidobacterium lactis BI-04


Lactobacillus acidophilus La-14

Lactobacillus salivarius Ls-33

Lactobacillus paracasei Lpc-37

Best Probiotic

Prescript-Assist

Reuteri

Align Bifidobacterium infantis 35624

Culture-dependent Analysis

Product Single Colony

Isolates Align 2

Best 36

Bio-Kult 34

Culturelle kids 2

DDS Acidophilus 4

Garden of Life Primal Defense HSO 1

Garden of Life Primal Defense Ultra 30

Hammer Nutrition Digest 4

Jarro-Dophilus Original 23

Nature's Bounty 2

Now Probiotic-10 47

Primadophilus Reuteri 12

Probiotic15 24

Raw Probiotics Women 40

261

Species Single Colony

Isolates Bacillus subtilis 8

Bifidobacterium animalis 12

Bifidobacterium longum 6

Lactobacillus acidophilus 57

Lactobacillus brevis 3

Lactobacillus casei group 34

Lactobacillus fermentum 3

Lactobacillus helveticus 12

Lactobacillus plantarum 60

Lactobacillus reuteri 16

Lactobacillus rhamnosus 20

Lactobacillus salivarius 23

Lactobacillus zeae 5

Streptococcus thermophilus 2

261

PRIMADOPHILUS REUTERI

Serving size 1 capsule



Lactobacillus reuteri HA-188

5 billion

Capsule diluted in 5 ml saline

Serial 10-fold dilutions in saline and plating in MRS Agar

48 h incubation

Aerobiosis at 30 °C few very small colonies

Aerobiosis at 37 °C 1.1 x 109

CO₂ system at 37 °C 1.3 x 109

Campy system at 37 °C 1.3 x 109

Anaerobiosis 37 °C 1.4 x 109 per capsule

Assembly Strain K-mer Coverage >20x Total contigs

Total size of

contigs Longest contig

N50

contig

length

R1 Lactobacillus rhamnosus 99.88 314x 371 2891222 397843 175159

R2 Lactobacillus rhamnosus 99.81 318x 593 2895284 261490 128009

R3 Lactobacillus helveticus 99.39 717x 462 2030935 103617 26069


R5 Lactobacillus reuteri 99.46 169x 426 2109732 171310 59944

R6 Lactobacillus reuteri 99 200x 1214 2095234 195587 56636





High quality sequences of the microbial genomes

that allow for antibiotic resistance, virulence

factors and toxin genes characterization

NOW PROBIOTIC-10

Serving size 1 capsule

Lactobacillus acidophilus La-14 Bifidobacterium lactis BI-04

Lactobacillus plantarum Lp-115 Bifidobacterium longum BI-05

Lactobacillus rhamnosus Lr-32 Streptococcus thermophilus St-21

Lactobacillus paracasei Lpc-37 Bifidobacterium breve Bb-03

Lactobacillus salivarius Ls-33 Lactobacillus casei Lc-11

50 billion

Capsule diluted in 5 ml saline

Serial 10-fold dilutions in saline and plating in MRS Agar

48 h incubation



CO2 system at 37 °C 3.1 x 1010

Campy system at 37 °C 3.3 x 1010

Anaerobiosis 37 °C 3.3 x 1010

Assembly Strain K-mer Coverage >20x Total contigs

Total size of

contigs Longest contig

N50

contig

length

P10-29 Lactobacillus salivarius 99.9 54x 94 1983752 161875 56969

P10-3 Lactobacillus acidophilus 99.9 200x 50 1969979 405017 113236

P10-30 Bifidobacterium animalis 99.5 95x 67 1918987 219509 68213

P10-35 Bifidobacterium animalis 99.8 145x 27 1920102 531273 200990

P10-4 Lactobacillus plantarum 99.0 130x 70 3217239 484131 156849


P10-6 Lactobacillus plantarum 99.0 155x 66 3218223 269017 101425


P10-9 Lactobacillus acidophilus 99.9 160x 52 1969504 386684 120761

MISSING

Lactobacillus rhamnosus Lr-32

Lactobacillus paracasei Lpc-37

Bifidobacterium longum BI-05

Streptococcus thermophilus St-21

Bifidobacterium breve Bb-03

Lactobacillus casei Lc-11

CP000033 NCFM Commercially strain used for formula, yogurt, and fluid milk production

CP005926 La-14 DuPont

CP010432 FSI4 Fidelity Systems, Inc

AYUB01 CFH 50 cells from yogurt starter culture

Phylogenic relationships among

Lactobacillus acidophilus strains

CP

00

00

33

CP

00

59

26

CP

01

04

32

AY

UB

01

B1

4

DD

S4

HD

3

JO1

NB

2

P1

0-1

PD

U1

0

CP000033 0 70 68 73 68 68 69 68 70 68 66

CP005926 70 0 8 3 4 4 5 4 6 4 12

CP010432 68 8 0 11 11 11 12 11 13 11 3

AYUB01 73 3 11 0 6 6 7 6 8 6 14

B14 68 4 11 6 0 0 1 0 2 0 8

DDS4 68 4 11 6 0 0 1 0 2 0 8

HD3 69 5 12 7 1 1 0 1 3 1 9

JO1 68 4 11 6 0 0 1 0 2 0 8

NB2 70 6 13 8 2 2 3 2 0 2 10

P10-1 68 4 11 6 0 0 1 0 2 0 8

PDU10 66 12 3 14 8 8 9 8 10 8 0


Lactobacillus reuteri strains

CP

00

07

05

AP

00

72

81

CP

01

10

24

AC

LB0

1

AZD

D0

1

AA

PZ0

2

R7

_S2

Raw

-26

CP000705 0 8 3 11 7 42895 41235 7

AP007281 8 0 5 13 9 42897 41237 9

CP011024 3 5 0 8 4 42892 41232 4

ACLB01 11 13 8 0 10 42768 41096 12

AZDD01 7 9 4 10 0 42860 41198 8

AAPZ02 42895 42897 42892 42768 42860 0 16382 42307

R7_S2 41235 41237 41232 41096 41198 16382 0 40773

Raw-26 7 9 4 12 8 42307 40773 0

CP000705 DSM20016 US DOE Joint Genome Institute

AP007281 JCM1112 Kitasato Institute for Life Sciences

CP011024 IRT IBS and Pohang University of Science and Technology

ACLB01 MM2-3 Baylor College of Medicine

AZDD01 DSM20016 Intestine of adult

AAPZ02 100-23 DOE Joint Genome Institute

FM1

79

32

2

AP

01

15

48

AZC

Q0

1

AG

KC

01

JYC

S01

BA

LT0

1

Raw

-16

R2

FM179322 0 3 43254 43268 42830 44016 44250 43564

AP011548 3 0 43251 43265 42827 44014 44247 43561

AZCQ01 43254 43251 0 41601 41120 16 9 42043

AGKC01 43268 43265 41601 0 13 42157 42337 196

JYCS01 42830 42827 41120 13 0 41675 41855 207

BALT01 44016 44014 16 42157 41675 0 17 42619

Raw-16 44250 44247 9 42337 41855 17 0 42782

R2 43564 43561 42043 196 207 42619 42782 0

FM179322 GG Institute of Biotechnology, University of Helsinki

BALT01 DSM 20021 = JCM 1136 =NBRC 3425 University of Tokyo.

AZCQ01 DSM 20021 = JCM 1136 =NBRC 3425 Inner Mongolia Agricultural University,1989

AGKC01 R0011 dairy starter culture, Canada

JYCS01 CLS17 stool sample, Kazakhstan: Astana


Lactobacillus rhamnosus strains

0.25%

afaD aggR bfpA eae pap saa pINV

Adhesins

cdt cnf ehx hly LT/ST stx sub

Toxins

310 Representative SNP Genotypes

% A B1 S2 S3 E2 E D F1 F2 B2 CL1

A 1.1 1.4 1.3 1.2 1.4 2.0 2.1 2.1 2.3 3.2

B1 1.1 1.3 1.2 1.3 1.4 2.0 2.1 2.1 2.3 3.2

S2 1.4 1.3 1.3 1.4 1.5 2.0 2.1 2.1 2.3 3.3

S3 1.3 1.2 1.3 1.5 1.5 2.1 2.2 2.2 2.4 3.3

E2 1.2 1.3 1.4 1.5 1.5 2.0 2.1 2.1 2.3 3.2

E 1.4 1.4 1.5 1.5 1.5 2.0 2.1 2.1 2.3 3.2

D 2.0 2.0 2.0 2.1 2.0 2.0 1.8 1.7 2.2 3.0

F1 2.1 2.1 2.1 2.2 2.1 2.1 1.8 1.8 1.9 3.0

F2 2.1 2.1 2.1 2.2 2.1 2.1 1.7 1.8 1.8 3.1

B2 2.3 2.3 2.3 2.4 2.3 2.3 2.2 1.9 1.8 3.3

CL1 3.2 3.2 3.3 3.3 3.2 3.2 3.0 3.0 3.1 3.3

A

B1

B2

D

E F1

F2

Shigella

Escherichia sp. CL1

S1

S2 S3

SS

SD1

E2

Metagenomics: Shift in Perspectives and

Applications

Culture Independent Diagnostics

• Essentially probing metagenomes

• Can take reductionist approach

with wealth of whole genome

sequences

Platforms

• Whole genome sequencing

• Microarray

Considerations

• Sensitivity

• Specificity

Product

culture-independent

metagenomics

sequence

K-mer analysis

Cosmos ID

MetaPhlAn

Identification

Direct-from-Product DNA Isolation and

Sequencing

• Cell lysis and DNA exposure occurs by mechanical and

chemical methods

• Whole genome shotgun sequencing

Pipeline for K-mer database

Selected Reference Genomes

565 Bacterial genomes representing 100 different

species

• Match reads against the k-mers • Assign, Count, Normalized reads

Output Table of relative abundance per

species

25-mers specific for the different species were identified

DB: ~40-80K K-mers per species

Input Shotgun

Metagenome Reads (FastQ)

Metagenomic Analysis Detection Limits

Patro et al. mSphere 2016;1:e00057-16

Bifidobacterium longum



Bifidobacterium animalis

Lactobacillus plantarum

Lactobacillus casei group

Lactobacillus reuteri

Bifidobacterium bifidum

Target name Probe ct Read ct Reads

matched

Relactive %

Present

Bifidobacterium longum 32599 1316884 357935 32.286

Lactobacillus acidophilus 70469 1941608 445064 27.228

Lactobacillus rhamnosus 54371 2164056 278159 15.268

Bifidobacterium animalis 74717 1925751 171874 10.601

Lactobacillus plantarum 50527 2374868 155677 7.786

Lactobacillus casei group 60168 1964209 98467 5.955

Lactobacillus reuteri 32513 1146264 4888 0.507

Bifidobacterium bifidum 66219 1991129 3092 0.184

Bifidobacterium breve 56216 1787121 994 0.066

Lactobacillus iners 28902 1044277 206 0.023

Pediococcus acidilactici 26488 1173449 145 0.015

Lactobacillus pentosus 129105 3364781 347 0.012

Lactobacillus zeae 113644 2962158 259 0.010

Lactobacillus kefiranofaciens 66218 1837936 145 0.009

Lactobacillus helveticus 50002 1606399 107 0.008

Bifidobacterium dentium 97911 2601231 148 0.007

Bifidobacterium adolescentis 47337 1537553 62 0.005

Paenibacillus 1128 100000 3 0.004

Lactobacillus jensenii 6754 363487 10 0.003

Ruminococcus albus 2843 197757 5 0.003

Lactobacillus crispatus 42400 1365893 21 0.002

Lactobacillus sanfranciscensis 48974 1280377 17 0.002

Bifidobacterium asteroides 85360 2159745 28 0.002

Lactobacillus fermentum 72844 1990265 24 0.001

Clostridium difficile 63459 3088547 27 0.001

Lactobacillus brevis 88585 2255504 11 0.001

Lactobacillus salivarius 37452 1353277 6 0.001

Desulfovibrio vulgaris 69658 3084907 13 0.001

Bacillus cereus 37787 1976640 8 0.000

Enterococcus faecalis 79578 2446128 6 0.000

Faecalibacterium prausnitzii 7843 419474 1 0.000

Enterococcus faecium 84870 2432977 5 0.000

LABEL:

Bifidobacterium longum BB536

Lactobacillus acidophilus La-14


Lactobacillus rhamnosus R0011

Lactobacillus casei R0215

Lactobacillus plantarum R1012

Label authentication: Identification and relative quantification of microbial species directly from the product using K-mer analysis

Metagenomic sequencing of various

probiotic products and associated

inconsistences with labelling


The relative amounts of each bacteria in a product are reported for two examples, product G and Product H. The

variation in Project G is due to the different formulation in lot 1 versus lots 2 and 3 and can be detected via sequencing.

(Patro et al. 2016. mSphere, 1:e00057-16).

Lot to lot variation and contaminant identification

Bacterial Genera Representation

Lactobacillus

Bifidobacterium

Bacteroides

Bacillus

Lactococcus

Leuconostoc

Pediococcus

Clostridium

Enterococcus

Streptococcus

12%

41%

16%

6%

10%

12%

Custom “GutProbe” Microarray Design

10 Genera 92 Genomes 229 Plasmids

Validation

Validation

Probesets displayed for type strains Establishing correct annotation and cross-

hybridization is essential

Genome-scale Product and Strain

Comparison

Journal of Applied Microbiology

Volume 118, Issue 6, pages 1478-1488, 9 APR 2015 DOI: 10.1111/jam.12795

http://onlinelibrary.wiley.com/doi/10.1111/jam.12795/full#jam12795-fig-0004

http://onlinelibrary.wiley.com/doi/10.1111/jam.2015.118.issue-6/issuetoc







Primadophilus Intensive “active ingredients” B. Breve, B. longum, B. bifidum, L. acidophilus, L. rhamnosus

Bacillus cereus ATCC 10987 Bacillus coagulans 36D1

Bacillus pumilus SAFR-032 Bacillus subtilis subsp. subtilis str. 168

Bacteroides caccae ATCC 43185 Bacteroides capillosus ATCC 29799

Bacteroides cellulosilyticus DSM 14838 Bacteroides coprocola DSM 17136

Bacteroides coprophilus DSM 18228 Bacteroides dorei DSM 17855

Bacteroides fragilis NCTC 9343 Bacteroides intestinalis DSM 17393

Bacteroides stercoris ATCC 43183 Bacteroides thetaiotaomicron VPI-5482

Bacteroides uniformis ATCC 8492 Bifidobacterium adolescentis ATCC 15703

Bifidobacterium adolescentis L2-32 Bifidobacterium angulatum DSM 20098

Bifidobacterium bifidum NCIMB 41171 Bifidobacterium breve DSM 20213

Bifidobacterium catenulatum DSM 16992 Bifidobacterium dentium Bd1

Bifidobacterium gallicum DSM 20093 Bifidobacterium longum bv. infantis CCUG 52486

Bifidobacterium longum DJO10A Bifidobacterium longum DJO10A

Bifidobacterium longum NCC2705 Bifidobacterium longum subsp. infantis ATCC 15697

Bifidobacterium longum subsp. infantis ATCC 55813 Bifidobacterium pseudocatenulatum DSM 20438

Clostridium asparagiforme DSM 15981 Clostridium bolteae ATCC BAA-613

Clostridium hathewayi DSM 13479 Clostridium leptum DSM 753

Clostridium methylpentosum DSM 5476 Lactobacillus antri DSM 16041

Lactobacillus brevis ATCC 367 Lactobacillus buchneri ATCC 11577

Lactobacillus casei ATCC 334 Lactobacillus crispatus JV-V01

Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 Lactobacillus fermentum IFO 3956

Lactobacillus helveticus DPC 4571 Lactobacillus helveticus DSM 20075

Lactobacillus hilgardii ATCC 8290 Lactobacillus iners DSM 13335

Lactobacillus jensenii JV-V16 Lactobacillus johnsonii NCC 533

Lactobacillus paracasei subsp. paracasei ATCC 25302 Lactobacillus plantarum WCFS1

Lactobacillus rhamnosus GG Lactobacillus rhamnosus HN001

Lactobacillus rhamnosus Lc 705 Lactobacillus rhamnosus LMS2-1

Lactobacillus ruminis ATCC 25644 Lactobacillus sakei subsp. sakei 23K

Lactobacillus salivarius UCC118 Lactobacillus ultunensis DSM 16047

Lactococcus lactis Lactococcus lactis subsp. lactis

Leuconostoc citreum KM20 Pediococcus pentosaceus ATCC 25745

B. bifidum NCIMB 41171 2%

B. longum ATCC 15697 9% B. breve DSM 20213

60%

L. rhamnosus LMS2-1 3%

Batch formulations by two

different genomic-scale

techniques


Whole genome sequencing (WGS) can facilitate the review of NDIs:

Identity, safety, history of use => “molecular sleuthing”

Metagenomic sequencing offers high potential for post market

surveillance and labeling

• Sample complexity, detection limits

Microarray offers rapid and highly specific identification and

subtyping of marketed products => GMP monitoring

Enhanced databasing will improve species taxonomy, strain

identification, and provide new refined targets for applied tool

development

Conclusions/Summary

Acknowledgments

• CARMEN TARTERA

• JAYANTHI GANGIREDLA

• TAMMY BARNABA

• MARK MAMMEL

• DAVID LACHER

• JENNIFER PATRO

• PADMINI RAMACHANDRAN

• ERIKA PFEILER

• JADA LEWIS

Dwayne C. Savage, Ph.D.

1934 - 2016

• Pioneer • Educator • Eminent Commensal Microbiologist

Next-generation Toolbox for Probiotic Analysis · Next-generation Toolbox for Probiotic Analysis...

Documents

Transcript of Next-generation Toolbox for Probiotic Analysis · Next-generation Toolbox for Probiotic Analysis...