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Bacteriocins as alternatives to antibiotics : the case of enterocins DD14 AND DD28

OIE headquarters, Paris, FranceDecember 13th 2016

Pr. Djamel DRIDERLille University Sciences and Technologies

E-mail : djamel.drider@univ-lille1.frTwitter @DjamelDrider

I would like to thank- Dr. Cyril G. Gay (USDA)- Dr. Bruce Seal (Oregon State University)

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What are bacteriocins ?Bacteriocins are AMPs :

- ribosomally synthesized- proteinaceous nature- non-cytotoxic (usually)- active against bacteria which are closely related to the

producing strains but…- stable at extreme pHs and temperatures- produced by both Gram-negative and Gram-positive bacteria

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> 2756 AMPs registered in APDhttp://aps.unmc.edu/AP/main.php

Nebraska University – USA.

- 276 Bacteriocins- 337 Plants- 04 Archaea- 08 Protists- 13 Fungi- 2070 Animals

Antimicrobial Peptides Database content on December, 12th 2016

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BACTERIOCINS OR REAL SWISS KNIFE

Functions allocated to bacteriocins- Antimicrobial Agents- Antibiotics-Improving Complementary Agents- Antiviral Agents- Microbiota Regulators- Anticancer Agents- Plant Growth Promotors- Emerging functions (immuno-stimulating agents, virulence factor)

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BACTERIOCINS

Gram-negative bacteriaEnterobacteriaceae Bacilli

Lactic acid bacteriaColicins

Microcins (> 40 representatives) Bacteriocins – hundreds -

COMMON POINTSStability at extreme pHs and temperatures

MW < 10 kDa

Microcins (class I) 1 < MW < 3 KDa Post-translational modifications

(lasso-peptides)

Microcins (classe II) 6 < MW < 10 KDaSiderophores peptides

Gram-positive bacteria

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Mode of action of LAB bacteriocins

Pore forming Cell – membrane target

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MODE OF ACTION THROUGH PORE-FORMING

Intracellularside K+, amino-acids, inorganic phosphate, ATP

+ --+

Extracellularside

8NAGHMOUCHI, DRIDER and FLISS. (2007). Journal of Applied Microbiology 102:1508-1517.

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Cell-membrane targetFigure two

Lipid II : Concentration “nanomolar”

Figure credit Dr. Paul CotterUniversity college, Cork. Ireland

Class IIa bacteriocin

Interaction with docking protein on the cell membrane (MptC)

2. Pore-formation andpermeabilization of the cellmembrane

Figure from Drider et al. Mic Mol Biol Reviews (2006)

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uracil phosphoribosyl transferase

AnimalsHumans

LAB bacteriocins inputs – Management of antibiotics

- Replacement of fading antibiotics (in some cases !)- Antibiotics-Improving Complementary Agents ! (potentialization of antibiotics)

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- 6 samples of meconium from six blind donors (Roubaix hospital, France) - Isolation on MRS medium- Selection of Gram-positive and devoid of catalase activity (Lactic Acid Bacteria)

Distribution of LAB isolates

107 bacterial isolates

0

5

10

15

20

25

30

1 2 3 4 5 6

num

bers

of b

acte

ria in

sam

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Samples

20 20

12 12

26

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Identification of bacterial isolatesClassical and molecular methods

Enterococcus faecalis

141414

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10

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1 2 3 4 5 6

num

bers

of b

acte

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sam

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Samples

(Ef 14, Ef 28, Ef 90, Ef 97 and Ef 101) (Ef 93)

Ef 14

Ef 28 Ef 90

Ef 93

Ef 97Ef 101

Antimicrobial activity by welldiffusion method against Listeria

innocua

Screening of antibacterial activity

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Indicator strains Ef 14 Ef 28 Ef 90 Ef 93 Ef 97 Ef 101

BN AN BN AN BN AN BN AN BN AN BN ANListeria monocytogenes +++* ++ +++ ++ ++ ++ +++ ++ +++ ++ +++ +Listeria innocua F +++ +++ +++ + ++ ++ ++ ++ +++ ++ +++ ++Bacillus subtilis +++ +++ +++ ++ +++ ++ +++ ++ ++ + +++ ++Staphylococcus aureus ATCC33862

++* ++ ++ ++ +++ ++ ++ ++ ++ ++ ++ ++Staphylococcus aureus (MRSA) +* - ++ - + - + - + - + -Enterococcus faecalis +++ +++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++E. coli CIPI103982 +++ - ++ - ++ - ++ - ++ - ++ -Klebisella oxytoca +++ - ++ - ++ - ++ + ++ - ++ +Proteus mirabilis ++ - ++ - +++ - + - ++ - ++ -Salmonella heidbelberg ++ - ++ - ++ - ++ - ++ - ++ -Pseudomonas fluorescens

++ - ++ - ++ ++ ++ - ++ - +++ +Candida albicans - - - - - - - - - - - -Saccharomyces cerevisiae

- - - - - - - - - - - -

SPECTRUM OF THE ANTAGONISTIC STRAINS

* : + (1-3 mm), ++ (3-6 mm), +++ ( > 6 mm)

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S. aureus ATCC 33862 vs E. faecalis 28

S. aureus ATCC 33862 vs E. faecalis 93

S. aureus ATCC 33862 vs E. faecalis ATCC 29212

- Drop in S. aureus ATCC 33862 counts in the presence of E. faecalis 28 or E. faecalis 93- Stability in S. aureus ATCC 33862 counts in the presence of E. faecalis ATCC 29212

CHALLENGE TESTS S. aureus vs. E. faecalis

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DD28

DD93

MW= 5204.21 Da

MW= 5203.89 Da

Purification of enterocins DD28 and DD93

Mass spectrometryRP-HPLC of Enterocins DD28 and DD93

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Antibiotics MIC (mg/L) Antibiotics MIC (mg/L)

Benzylpenicillin R 0.25 Linezolid S 2Oxacillin R 1 Teicoplanin S ≤ 0.5Gentamicin S ≤ 0.5 Vancomycin S 1Kanamycin R 32 Tetracyclin S ≤ 1Tobramycin R ≥ 16 Fosfomycin S ≤ 8Ofloxacin S ≤ 0.5 Nitrofurantoin S ≤ 16Erythromycin R ≥ 8 Fusidic acid S ≤ 0.5Lincomycin R ≥ 16 Rifampin S ≤ 0.03Pristinamycin S 1 Trimethoprim-sulfamethoxazole S ≤ 10

Antibiotic susceptibility of MRSA-S1 (clinical isolate)

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Effects of antimicrobials combinations against MRSA-S1Ch

ecke

rboa

rd as

says

Kill-

curv

es ex

perim

ents

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Effect of antimicrobials on MRSA-S1 biofilm formation on AISI 304L stainless steel

Epifluorescence microscopy

Time (h) 0h 3h 6h 24h

Control 5.55±0.08 7.25±0.15 7.81±0.18 8.85±0.11

Erythromycin (8 µg/ml) 5.25±0.25 7.09±0.19 7.39±0.29 8.57±0.12

Ofloxacin (0.5 µg/ml) 3.12±0.12 5.24±0.22 5.58±0.18 6.45±0.13

Vancomycin (1 µg/ml) 3.10±0.08 5.29±0.19 5.20±0.14 6.10±0.03

Rifampin (0.03 µg/ml) 3.11±0.10 5.17±0.11 5.28±0.17 6.06±0.04

Enterocin DD28 (200 µg/ml) 3.29±0.1 5.51±0.1 5.62±0.13 6.54±0.12

Enterocin DD93(200 µg/ml) 3.35±0.21 5.59±0.1 5.58±0.19 6.58±0.17

Erythromycin / DD28 (1/50 µg/ml) 3.35±0.1 5.32±0.17 5.15±0.25 6.13±0.1

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SEM of biofilms formation by MRSA-S1 on glass slides

CVancomycin

A Control B DD28

D DD28 + Erythromycin

Key information

Enterocins DD28 and DD93 but also DD14, DD90, DD97 and DD101 displayed 100% identical sequences to enterocin 7 (strain isolated from meat in Canada) and enterocin MR10 (strain isolated from bird in Spain)

The resulting sequences were blasted on blastn pubmed database and showed complete alignment with the sequences of enterocin MR10A and MR10B

International Journal of Antimicrobial Agents (In press)

The French National Research Agency Projects for scienceProject SINCOLISTIN (2015-2018).

Phileo Lesaffre Animal CareProjects Clostrisaf I and Clostrisaf II

Financial supports

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Thank you for your attention