DAIRY MICROFLORA: LESSONS FROM METAGENOMICS

Denis Roy*, Gisèle LaPointe, Geneviève Gagné and Émilie Desfossés-Foucault

Université Laval

Colloque STELA 2013 / STELA Symposium 2013 13 mai 2013 Hotel Delta Centre-Ville, Montréal

• Metagenomics: definition/methods • High-throughput sequencing: dairy

microflora • Dynamics of lactococci and lactobacilli in

cheedar cheese • Activity of lactococci and lactobacilli in

cheedar cheese

Summary

“Detectomics!”

Dairy microbiologist s are unlikely to be able without detectomics; a major priority Develop and optimize molecular methods for:

• Detection • Reliable identification • Monitoring and determination

Microorganisms in dairy environment

Ecolini D 2013. High-throughput sequencing and metagenomics: a step in the culture independent analysis of food microbial ecology. Appl Environ Microbiol.

Farm /Plant Cheese Starters/ Cultures

Community genomics

Meta-genomics or environmental genomics

• The discipline that uses genomic methods to analyze the natural ecological communities.

Metagenomics

• Sequencing and analysis of DNA of microorganisms recovered from a given environment

“Cheese-omics”

• Various methods to study genomic diversity, activity and metabolism of the various populations of the cheese ecosystem without cultivate them

Influence of microbial population

Diversity Dynamics Activity

5

“*-Omics”

DNA : Total population RNA : Active population

Milk or cheese sample

Culture DNA/RNA Extraction

qPCR PCR amplification of the 16S rRNA

Separation of 16S rRNA by gel

electrophoresis (DGGE, T-RFLP,

ARISA)

Sequencing of cloned 16S rRNA amplicons

Direct sequencing of 16S rRNA amplicons by Pyrosequencing (Roche 454)

Overview of molecular techniques

+ -

High-throughput sequencing

HTS

MILK

Strain monitoring

Microbiota

Microbiome

Sequencing of the 16S rRNA

Dairy samples

Variable region

Amplicon length

Sequencing platform

Taxonomic resolution

Reference

Bovine Mastitic milk

V1-V2 250 bp 454 FLX Family/genus Oikinomou et al., PloS one. 2012;7.

Danish raw milk / cheese

V3-V4 450 bp 454 FLX Genus/species Masoud et al. 2011. Int Dairy J 21,142 Masoud et al. 2012 Int J Food Microbiol 153, 192

Water buffalo Mozzarella cheese

V1-V3 500 bp 454 FLX

Species Ercolini et al. 2012, Appl Environ Microbiol 78:8142

Irish artisanal cheese

V4 250 bp 454 FLX Genus Quigley et al. 2012. Appl Environ Microbiol 78, 5717

HTS in dairy samples targeting the 16S rRNA gene

Oikonomou G, Machado VS, Santisteban C, Schukken YH, et al. (2012) Microbial Diversity of Bovine Mastitic Milk as Described by Pyrosequencing of Metagenomic 16s rDNA. PLoS ONE 7(10): e47671.

Streptococcus

Staphylococcus

Healthy

Propionibacterium

Lactobacillus

Strep Staph

Microbial diversity of Bovine Mastitic Milk by pyrosequencing of Metagenomics 16s rDNA

Day 3 Day 7 UT CO2 TH MF UT CO2 TH MF

R2J3C4 F7

R4J3L4 F5

R2J7T4 D3

R2J3L4 A7

R2J3C4 A6

R2J3L4 F6

R2J7C4 B1

R2J3L4 B9

R4J3C4 B1

R4J3L4 A12

R2J3L4 A9

R2J3L4 F8

R2J3T4 B10

R2J3L4 B4

R4J3L4 H11

R2J3T4 C5

R2J7T4 G4

R2J3L4 H6

R2J3T4 D10

R2J7T4 E3

R2J7T4 H4

R2J3L4 F5

R2J3L4 A11

R4J3C4 B11

R2J3L4 H5

R2J3L4 D3

R2J3L4 C6

R4J3T4 C12

R2J3C4 E12

R2J3L4 A1

R2J3L4 B5

R2J3L4 D1

R2J3C4 B6

R2J7M4 C6

R4J7T4 A12

R2J3C4 G1

R2J3T4 F8

R2J3C4 D10

R4J3C4 G1

R2J3L4 A8

R4J3L4 H2

R2J3L4 B2

R2J3L4 E3

R2J3L4 F3

R2J3T4 F12

R4J3L4 D1

R4J3T4 C2

R2J3C4 C7

R4J3M4 G2

R2J7T4 E4

R2J3C4 C11

R4J3C4 H12

R2J7M4 G10

R2J7M4 C12

R2J3L4 G10

R2J3C4 H9

R2J3L4 B12

R2J3C4 F11

R2J3T4 C11

R2J3C4 D2

R2J3C4 B5

R2J3T4 A9

R2J7M4 F11

R2J7M4 D12

R2J3C4 E6

R2J3T4 H4

R4J3M4 E10

R4J7C4 H11

R2J3L4 A3

R4J3L4 D9

R2J3T4 F9

R2J3L4 H7

R4J3C4 E1

R2J7T4 H10

R2J3C4 E9

R2J3L4 B11

R2J3T4 B7

R4J3L4 E3

R4J3L4 C6

R2J3L4 G7

R2J3L4 H11

R2J7M4 A3

R2J3C4 A2

R4J7T4 B11

R2J3M4 H9

R2J7M4 G8

R2J3C4 E7

R2J3T4 D1

R2J3T4 A8

R2J3M4 A12

R2J7M4 E5

R4J3C4 G11

R2J7T4 D6

R2J3L4 G1

R4J3T4 E8

R4J3T4 E11

R2J3C4 G6

R4J3L4 E9

R4J3T4 H5

R2J7T4 B8

R2J3C4 H10

R2J3C4 D3

R2J7T4 B12

R2J3T4 G1

R2J3L4 D6

R2J3T4 H1

R2J3C4 F6

R2J3M4 E9

R2J7M4 D11

R2J3C4 A3

R4J7T4 G12

R2J3L4 D11

R2J3L4 B6

R2J3L4 C3

R4J3M4 A3

R2J3M4 D5

R2J3L4 E1

R2J3L4 G9

R2J3C4 E5

R2J3L4 B1

R2J3L4 G2

R2J3L4 G4

R2J3L4 F4

R2J3C4 H3

R2J7T4 C2

R4J7T4 H9

R2J3T4 G4

R2J3L4 G12

R4J3C4 B2

R4J3M4 A5

R4J7M4 C1

R2J3L4 B10

R2J7T4 B2

R2J3L4 E2

R4J3T4 C10

R2J3L4 H4

R2J7T4 F5

R4J3L4 G7

R2J3M4 A1

R2J7T4 H7

R2J7T4 E8

R2J3T4 H10

R4J3M4 B1

R2J3M4 G3

R2J3M4 C9

R4J7C4 G1

R2J3L4 C4

R2J3T4 E1

R4J3C4 H9

R4J7C4 C4

R4J3C4 G4

R2J3T4 C2

R2J7T4 B5

R2J3M4 B2

R2J3T4 E6

R2J3M4 H1

R2J7M4 D3

R2J3M4 B11

R2J7T4 D4

R4J3M4 E3

R4J3L4 C9

R4J3C4 D7

R4J3M4 E12

R2J3C4 H4

R2J3M4 C2

R2J3M4 C5

R2J3L4 C5

R2J7M4 F5

R2J3M4 D11

R2J3L4 G8

R4J3M4 B3

R2J3L4 F2

R2J7L4 B3

R2J3M4 D1

R2J3M4 F9

R2J3M4 B1

R2J3L4 H2

R4J3L4 D6

R4J3M4 F1

R2J3L4 A4

R2J3T4 H6

R2J3L4 C2

R4J3M4 G6

R2J7T4 H2

R4J3C4 H3

R4J3M4 H9

R4J3L4 F1

R2J3C4 F5

R2J7T4 C9

R2J3C4 H12

R2J7M4 G9

R4J3C4 B8

R4J3C4 C8

R2J3T4 G9

R2J7M4 F3

R2J3C4 C2

R4J3T4 D8

R4J3C4 D11

R2J3C4 B9

R2J3T4 C6

R2J3C4 E10

R2J3T4 C8

99

99

99

99

99

97

85

64

99

79

99

99

99

86

47

99

99

99

99

94

73

45

40

32

28

22

15

99

99

99

99

99

99

75

62

92

99

99

99

99

99

99

44

99

86

52

85

99

98

99

95

99

99

92

99

99

98

99

35

50

88

72

99

99

98

98

93

89

86

65

61

43

23

20

25

98

52

69

86

98

63

47

97

96

96

79

82

58

70

68

96

62

78

64

94

92

92

64

82

56

89

91

90

76

52

89

84

82

88

77

34

85

80

83

77

65

73

72

58

57

44

41

57

30

54

41

53

49

49

39

25

24

18

45

87

47

77

71

62

52

50

54

79

87

50

86

76

92

62

50

58

42

42

21

41

28

24

22

20

19

12

11

10

12

24

43

69

92

95

93

77

73

72

50

69

51

63

54

52

98

33

33

19

22

26

84

0.02

Bacilli Clostridia

Actinobacteria Proteobacteria

Bacteriodetes

Ochrobactrum

Lysinibacillus

Enterococcus

Corynebacterium

Clostridium

Facklamia

Acinetobacter

Pseudomonas

Stenotrophomonas

Delftia

Aerococcus

Trichochoccus

Streptococcus

Staphylococcus

Microbacterium

1 à 23 à 45 à 1011 à 2526 à 75>75

Number of sequence in

OTU Rasolofo E, St-Gelais D, LaPointe G, Roy D. Molecular analysis of bacterial population structure and dynamics during cold storage of untreated and treated milk. International journal of food microbiology. 2010;138:108-18.

Phylogenetic architecture of the two major classes of 16S rDNA clone libraries and their abundance in untreated, CO2, thermized and microltered milk samples during 7 d of storage at 4 °C.

Staphylococcus, Streptococcus, Clostridia, Aerococcus, Facklamia, Corynebacterium, Acinetobacter, and Trichococcus

Raw milk

Streptococcus

Enterococcus Facklamia

Aerococcus Acinetobacter

Corynebacterium

Staphylococcus

Masoud et al. 2012 Int J Food Microbiol 153, 192

Pseudomonas

Streptococcus thermophilus, Lactococcus lactis, Staphyloccus saprophyticus, Lactobacillus rhamnosus, Brevibacterium linens, Corynebacterium casei, Acinetobacter

Danish raw milk cheese

DNA

cDNA

Streptococcus, Lactococcus and

Lactobacillus

95-99% Lb. helveticus Rest: Lb. casei, Lb. delbrueckii and Lb.

fermentum

Staphylococcus

Metabolically active

Masoud et al. Characterization of bacterial populations in Danish raw milk cheeses made with different starter cultures by denaturating gradient gel electrophoresis and pyrosequencing. International Dairy Journal. 2011;21.

Ercolini D et al. Appl. Environ. Microbiol. 2012;78:8142-8145

Raw Milk Raw Milk

Streptococcus macedonicus Pseudomonas Acinetobacter 192 OTUs

16S rRNA gene pyrosequencing analysis of two mozzarella cheese batches

Ercolini D et al. Appl. Environ. Microbiol. 2012;78:8142-8145

Streptococcus

Clostridia

Acinetobacter

Corynebacterium

Pseudomonas

Bacterial diversity and relative abundance of two mozzarella cheese batches

Quigley L et al. Appl. Environ. Microbiol. 2012;78:5717-5723

Assignment of cheese microbiota at the genus level (a) soft cheese; (b) semihard cheese; (c) hard cheese; (d) cheese rinds

Irish artisanal cheese

21 8

Streptococcus Facklamia

Corynebacterium

Staphylococcus Pseudomonas 2

Rind

Influence of microbial population

Diversity Dynamics Activity

18

“*-Omics”

Presence : DNA : Total population (viable, dead or viable but not culturable (VNC)) Trancriptomic activity: cDNA : Active population

8

9

10

11

12

13

0 1 2 3 4 5 68

9

10

11

12

13

0 1 2 3 4 5 6

8

9

10

11

12

13

0 1 2 3 4 5 68

9

10

11

12

13

0 1 2 3 4 5 6

Log

geno

me

copy

nu

mbe

r/g o

f che

ese

Log

cDN

A co

py

num

ber/g

of c

hees

e

Ripening time (months)

SE

SE

SE

SE

Dynamics of lactococci

Pasteurized Themized

Total LAB

Lactococci

Cheddar Cheese

SE SE

SE SE

SE SE

Log

cDN

A co

py n

umbe

r/g o

f che

ese

456789

1011

0 1 2 3 4 5 6

456789

1011

0 1 2 3 4 5 6

456789

1011

0 1 2 3 4 5 6

456789

1011

0 1 2 3 4 5 6

456789

1011

0 1 2 3 4 5 6

456789

1011

0 1 2 3 4 5 6

Ripening time (months)

Dynamics of lactobacilli

Pasteurized Themized

Lactobacilli

Lb. casei/ paracasei

4 °C

7 °C

12 °C

Cheddar Cheese

Activity: Models

Inoculation

Cheesemaking : Pearce test

T0

T4 Salting T

5

Temps

Tem

péra

ture

T2 38 °C T3

35 °C

Ripening: slurry

Cheese models

T1 32 °C

109 cfu/g SK11

106 cfu/g 334

109 cfu/g SK11

106 cfu/g 334

X 3 rep

Lactococcus lactis ssp. cremoris SK11

Lactobacillus casei ATCC 334

Preculture in milk

(16h at 22 °C)

25 g of cheese powder(lfreeze-dried and irradiated)

Ripening for 3, 6, 9 et 12 days at 30 °C

Homogeneization with bacteria and nitrogen flush

RT-qPCR HT

Classification Genes Métabolisme des sucres deoC, pfk, adh, ldh, gapA, serA, galK

Métabolisme des a.a. cysK, metC, argH, aspB, hisC, glmS, nifS, glyA, luxS

Dégradation (a.a. et peptides) bcaT, araT, oppD, prtP, pepM, pepN, pepQ, pepX

Réponse au stress clpC, dnaJ, groEL, groES, htrA

Fonctions cellulaires dnaG, ftsE, dacA, purD

Lipolyse estA

Gènes spécifiques à SK11 acpD, busAA, cysE, holin, optD, argD, serC, trpG

Gènes spécifiques à ATCC 334 ycjM, aldB, aspA, fpb, gdh, lacG, opuA, lipA Relative

quantification (versus T0)

RNA extraction - cDNA

cDNA

L. Lactis ssp. cremoris SK11

A

Pear

ce

Slur

ry

Mixed

estA gene (lipolysis) up-regulated after salting

Up-regulation of cysK, aspB et hisC (amino acids metabolisms)

Up-regulation of prtP (proteinase) at the beginning of ripening

Up-regulation of ldh, adh, gapA et pepM, pepN at the end of ripening

Up-regulation of grosES (chaperone proteine) in single and mixed culture.

Up-regulation of galK, serA (sugar metabolism) and ftsE (cellular division).

L. casei ATCC 334

B

Up-regulation of ftsE (cellular division) during cheesemaking and ripening Pu

re

Mix

ed

Up-regulation of fbp (fructose 1,6 biphosphatase).

Up-regulation of metC in slurry

Up-regulation of pepQ, pepM and pepX at the end of ripening

Active non culturable

Viable Membrane compromised

VNC ghost

Culturable

Metabolically active

Intact

Propidium monoazide

Membrane: intact

Membrane : compromised

PMA

Photoactivation

I

II

III

IV PCR amplification

Monitoring of lactococcal strains

Pearce / autolytic Test

PMA qPCR

qPCR

Log

gene

cop

y nu

mbe

r/m

l

Tracking

Quality control (monitoring)

Process innovation

Perspectives

Thanks

28

All collaborators , students, researchers and trainees