Lactic Acid Bacteria against [i]Staphylococcus aureus[/i] in the milk production chain: applications from farm to fork

(1)

HAL Id: hal-01349544

https://hal.archives-ouvertes.fr/hal-01349544

Submitted on 5 Jun 2020

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Lactic Acid Bacteria against [i]Staphylococcus aureus[/i]

in the milk production chain: applications from farm to fork

Yves Le Loir

To cite this version:

Yves Le Loir. Lactic Acid Bacteria against [i]Staphylococcus aureus[/i] in the milk production chain:

applications from farm to fork. Séminaire de l’Université Laval, May 2016, Québec, Canada. �hal- 01349544�

(2)

Lactic Acid Bacteria against Staphylococcus aureus in the milk production chain:

applications from farm to fork

Yves LE LOIR UMR1253 STLO

Séminaire Université Laval , Québec

(3)

.02

OUTLINE

1. A few words about…

2. Staphylococcus aureus 3. Lactic acid bacteria

4. Staphylococcus aureus, as a « dairy pathogen » 5. LAB to solve the problem?

6. LAB against S. aureus / food safety

7. LAB against S. aureus / animal health

8. Conclusion

(4)

_01

A few words about…

Séminaire .03 Université Laval , Québec

(5)

.04

UMR1253 STLO

INRA Agrocampus Ouest France

www6.rennes.inra.fr/stlo/UMR-STLO

[email protected]

.

(6)

100,000 dairy farms 22 billions of liters 200,000 direct jobs

700 factories

60,000 employees

Source: CNIEL-French Dairy Board

Milk production in France

(3.8 millions of dairy cows)

Treatment & transformation

Some economic aspects

(7)

.06

Dairy processing in Brittany Milk production in Brittany

• 20,000 producers

• 21% French milk collected (5 Mt):

~ production of Denmark or Ireland

• 45 plants

Major plants in size and volume of milk processed

• Butter, milk powder, Swiss-type cheese

(8)

Egg production

47 millions of laying hens 14 billions of shell eggs

Egg transformation

5 major companies 276,000 t liquid equivalent

36% French shell eggs

France :

1st European producer

19 %

49 %

11 %

[ 0 ; 1 [ [ 1 ; 5 [ [ 5 ; 20 [ [ 20 ; 50 [

[0 ; 1 [ [1 ; 5 [ [5 ; 20 [ [20 ; 50 [

43 % 9 %

10 % 5 %

(9)

.08

1comité de direction

6 équipes de recherche

2plateformes

1équipe d’appui

~130 personnes

78 permanents

~20 doctorants 7 cellules R&D

(10)

STLO: main research areas

Increase the knowledge on molecular and

supramolecular structure of milk and egg components

(casein micelle, fat globule, minor proteins)

Understand protein-protein, protein-lipid, protein-mineral interactions leading to expected technological and

bioactivity functionalities

(whey proteins, casein, fat globule and egg white proteins)

and understand their digestibility

(11)

.010

Characterize biological reactions

(enzymatic, microbial)

&

diffusion in food matrix

^(cheese)

Analyze transfers during technological processes

(membrane technologies, spray drying)

Characterize interactions between

bacteria/environment

(cheese, egg, gastrointestinal tract and mammary gland)

with regard to product quality, food safety, animal health and probiotic activity

STLO: main research areas

(12)

_02

Staphylococcus aureus

(13)

.012

S TAPHYLOCOCCUS AUREUS

Spherical bacterium (coccus). Gram-positive Grape-like clusters. Catalase+

Facultative anaerobe Resist lysozyme

Opportunistic pathogen.

Humans and animals are the primary reservoirs (nasal passages and throats and on the hair and skin of 50 percent or more of healthy individuals).

Causes numerous infections in humans and animals Major cause of nosocomial infections

Extensively studied (virulence factors, bacteria-host interactions, antibiotic resistance…)

Complete genome sequence for hundreds of S. aureus strains (human origin) Genome sequence of animal strains:

ET3-1 (RF122), Newbould305 (bovine) ED133, O11, O46 (ovine)

ED98 (poultry)

Le Maréchal et al., 2011. J. Bacteriol.

Bouchard et al., 2012. J. Bacteriol.

(14)

Wide range of infections…

… Multifactorial and

toxin-based infections

Staphylococcal enterotoxins

Exfoliative toxins TSST-1

Staphylococcal enterotoxins

After Wertheim et al., 2005 Lancet Infect Dis

(15)

.014

EXPRESSION OF THE VIRULENCE FACTORS REGULATED BY DIFFERENT (COOPERATIVE) SYSTEMS

accessory gene regulator (agr)

staphylococcal accessory regulator (sar) staphylococcal accessory gene expression (sae)

The expression of some toxin genes is controlled by the agr system (e.g. tst, seb, sec, sed).

Some other virulence genes are not controlled by agr (e.g. sea)

(16)

REGULATION OF VIRULENCE EXPRESSION THE AGR SYSTEM

After Novick and Geisinger, 2008. Annu. Rev. Genet.

(17)

.016

Post-exponential Exponential

time TEMPORAL REGULATION OF VIRULENCE

FACTORS

Growth

STAPHYLOCOCCUS AUREUS

(18)

Activation Inhibition

REGULATION OF VIRULENCE EXPRESSION THE AGR SYSTEM… AND OTHER SYSTEMS

(19)

_03

Lactic Acid Bacteria

Séminaire .018 Université Laval , Québec May 2016

(20)

LACTIC ACID BACTERIA

Group of Gram positive bacteria

Facultative anaerobes, non motile, catalase negative, and do not sporulate Common feature:

Ferment sugars into lactic acid and acidify media down to pH 3.5-4.5 LAB includ cocci

(Lactococcus, Streptococcus, Leuconostoc, Pediococcus, Enterococcus)

and bacilli

(Lactobacillus)

Other LAB genera: Aerococcus, Alloiococcus, Carnobacterium, Dolosigranulum, Globicatella, Lactosphaera, Oenococcus, Tetragenococcus, Vagococcus, and Weissella

Widely used in food fermentations

Found in vaginal, gut, and mammary ecosystems (bovine)

(21)

_04

Staphylococcus aureus, as a «dairy pathogen»

(22)

Animal Health

Mastitis

4896€/year for a herd of 100 dairy cows⁽¹⁾ (~200 M€/year in France)

Lower « cheesability »

⁽²⁾

Food Safety

SFP outbreaks

(1) Halasa et al., 2009. Livest. Sc.

(2) Le Maréchal et al., 2011. DST

S. aureus is also a major problem all along the milk production chain.

Huge economic loss

(23)

_05

LAB to solve the problem?

(24)

S. aureus mastitis difficult to cure and prone to recurrence

« Once a Staph cow, always a Staph cow »

Mastitis is the first cause of antibiotic use in bovine herds

Poorly efficient in eradicating S. aureus in the udder context

(cure rate for S. aureusmastitis often <15% in cows)

MRSA and other resistant strains are not a problem in mastitis treatment and milk production (until now)

(25)

.024

In France, ~1000 tons/year of antibiotics used in veterinary medicine, over the last decade.

~39 % ~22 % ~20 %

Social demand for the reduction of antibiotics in Vet Med

(26)

Dairy fermentations require a rich and balanced ecosystem

(27)

.026

HOST

MICROBIOTA PATHOGEN

The three components of a pathosystem

(28)

_06

LAB against S. aureus / Food safety

(29)

.028

HOST

Fermented Foodstuffs

(30)

S. aureus

Fermented Foodstuffs

MICROBIOTA

Le Loir et al., 2003. Genet. Mol. Res.

Charlier et al., 2009. Int J Food Microbiol.

Cretenet et al., 2011. Dairy Sci Technol

Some S. aureus are able to produce staphylococcal

enterotoxins

(31)

.030

S. aureus and Food poisoning outbreaks

S. aureus is the second cause of FP outbreaks in France.

Staphylococcal Food Poisoning is due to staphylococcal enterotoxins preformed in the foodstuff.

Symptoms of SFP are abdominal cramps, nausea, vomiting, sometimes followed by diarrhea.

In France, milk and dairy products are prevalent in SFP outbreaks.

S. aureus is the most frequently isolated contagious pathogen from raw

milk.

(32)

Growth

T°

NaCl

pH Nutrition

Microbiota

SE

pH NaCl Nutrients Temperature

Bacteriocins H₂O₂

Cretenet et al., 2011. Dairy Science & Technology.

(33)

.032

0 50 100 150 200 250

0 5 10 15 20 25 30

time (h)

hld expression

0 2 4 6 8 10 12 14 16 18

0 5 10 15 20 25 30

time (h)

AgrA expression

0 10 20 30 40 50 60 70 80 90

0 5 10 15 20 25 30

time (h) sarA expressionExpression RNAIII ExpressionagrAExpressionsarA

L. lactis impairs the expression of 2 major virulence regulation systems in S. aureus

Inhibition of virulence expression

L. lactis S. aureus

0 1 2 3 4 5 6 7 8

0 5 10 15 20 25 30

time (h)

sec4 expressionExpressionsec

Time (h) Time (h)

0 1 2 3 4 5 6 7 8 9

0 5 10 15 20 25 30

time (h)

sel expressionExpressionsel

L. lactis modulates SE gene expression in S. aureus

Even et al., 2009. Appl. Environ. Microbiol.

Cretenet et al., 2011. Environ . Microbiol. Reports

NABAB project

(34)

L. lactis LD61 alone S. aureus MW2 alone

S. aureus MW2 with L. lactis LD61

Kinetics of: Growth

Redox potential

Partial oxigen pressure

Redox potential is involved in the agr inhibition by L. lactis

L. lactis LD61 alone

S. aureus MW2 alone

S. aureus MW2 with L. lactis LD61

L. lactis LD61 alone

S. aureus MW2 alone

S. aureus MW2 with L. lactis LD61 Nouaile et al., 2014. AEM

(35)

.034 S. aureus alone

S. aureus in mix culture

Redox potential is involved in the agr inhibition by L. lactis

Constant pH6.6 Microaerophilic conditions

Constant pH6.6 Potassium ferricyanide*

Constant pH6.6 pO2 at 50%

*: 24 mM, used to adjust the redox potential (prevents the drop in redox potential)

(36)

How does it work in cheese?

Séminaire Université Laval , Québec

(37)

.036

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7

0 5 10 15 20 25 30

time (h)

sea expression

0 1 2 3 4 5 6 7 8

0 5 10 15 20 25 30

time (h)

sec4 expressionsecexpression

pure mixed

0 1 2 3 4 5 6 7 8 9

0 5 10 15 20 25 30

time (h)

sel expressionselexpressionseaexpression

CDM Cheese Matrix

agr dependant No secinduction in cheese matrix

Other regulation system?

agr independant

Phage physiology?

Different enterotoxin profiles in cheese matrix vs CDM

(38)

Conclusions

L. lactis is able to inhibit S. aureus virulence expression through the inhibition of the agr system.

Both acidification and redox potential are involved in the inhibition.

S. aureus agr expression is not induced in a cheese matrix.

The expression of agr dependent enterotoxin genes is inhibited, but

that of sea is not.

(39)

.038

_07

LAB against S. aureus / Animal health

Séminaire Université Laval , Québec May 2016

(40)

HOST

The three components of a pathosystem

(41)

.040

S. aureus Microbiota / LAB

cow

The three components of a pathosystem

(42)

Mammary Gland mammaire

Entry of S. aureus

1 2 3 4 5

6

Multiplication Adhesion Internalization Intracellular replication

Lysis Dissemination

Adhesion – Internalization

• Escape from immune response

• Contribute to the chronicity of the infection

Can LAB inhibit these first steps of the infection?

(43)

.042

Inhibition of S. aureus invasion into mammary epithelial cells by L. casei CIRM-BIA 667

Damien Bouchard PhD Thesis

(44)

PREVENTION

L. casei against S. aureus adhesion

L. casei against S. aureus internalization

Mammary epithelial cells (MAC-T)

LAB

L. casei CIRM-BIA 667

S. aureus RF122 / severe Newbould / chronic

(45)

.044

Inhibition of S. aureus adhesion by L. casei

Inhibition of S. aureus adhesion by L. casei CIRM-BIA667 in a strain dependent manner

L. casei : cell ratio

0 : 1 200 : 1 400 : 1 2000 : 1

+ S. aureus

100 : 1

Inhibitory conditions

adhesion

(46)

Inhibition of S. aureus internalization by L. casei

Inhibition of S. aureus RF122 and Newbould internalization by L. casei CIRM-BIA667

S. aureus alone

10 : 1 50 : 1 100 : 1

S. aureus : cell ratio

+ L. casei 2000 : 1

Inhibitory conditions

internalization

(47)

.046

Impact of different treatment on S. aureus adhesion

S. aureus + heat-killed

L. casei S. aureus S. aureus + live

L. casei

S. aureus + lactic acid

(pH 6,8)

Supernatant of L.

casei after 2h in cell medium

• Heat-killed L. casei inhibit S. aureus adhesion at the same rates as live L. casei

• No inhibition of adhesion with supernatant and acidified medium

(48)

Impact of different treatment on S. aureus internalization

• Heat-killed L. casei increase S. aureus internalization

• No inhibition of internalization with supernatant and acidified medium

S. aureus + heat-killed

L. casei S. aureus S. aureus + live

L. casei

S. aureus + lactic acid

(pH 6,8)

Supernatant of L.

casei after 2h in cell medium

(49)

.048

Intracellular survival of S. aureus and L. casei

L. casei S. aureus

• L. casei does not affect the kinetics of decay of internalized S. aureus

• L. casei internalizes into MAC-T and survives with a greater efficiency than S. aureus

(50)

Host cell structure and viability during infection

• Same density of cell monolayer

• No major modifications of cell structure

• Bacterial treatment does not affect cell viability

(trypan blue assay, MTT assay)

PHALLOIDINSYTO 9

10µm 10µm

Cells

Actin

Protein or ADN

(51)

.050

Conclusion

L. casei inhibits S. aureus adhesion in a strain dependent manner

L. casei inhibits S. aureus RF122 and Newbould internalization

Live L. casei are required to inhibit S. aureus internalization

L. casei internalizes into epithelial cells with a better survival rate than S. aureus

L. casei treatment does not affect cell structure and cell viability

1.

2.

3.

4.

5.

(52)

_08

Conclusion

(53)

.052

S. aureus LAB

Pathogens might “prefer” to be commensal or mutualistic and to live in an environment that is not pathological to the host.

LAB are able to inhibit S. aureus growth and or S. aureus virulence in

various contexts

New insights Balance of eubiosis / dysbiosis Balance of commensalism / pathogenicity New methods Treat and prevent disease. Bring microbial solutions to microbial problems

« mammary probiotics » or natural-killer starter LAB

(54)

J. Nicoli

B. Seridan (doc)

Y. Le Loir, S. Even, L. Rault

M. Cretenet, D. Bouchard (docs)

J. Schrenzel P. François D. Hernandez

(55)

.054 UMR1253 STLO

INRA Agrocampus Ouest France

www6.rennes.inra.fr/stlo/UMR-STLO

[email protected]