Biocontrol of Staphylococcus aureus in Dairy Products.

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Biocontrol of Staphylococcus aureus in Dairy Products.

Lynn El Haddad and Nour Ben Abdallah


Staphylococcus aureuso Gram-positiveo Mesophileo Facultative anaerobico Tolerates 20% of NaClSource: Wikipediao Commensal bacteriumo Pathogenic bacterium3


o Foodborne illnesso Bad hygieneo Ingestion of food containing >100 ngof staph enterotoxinso Mild to dangerous symptomso Responsible for rejecting batches ofraw milk4


o Solution envisioned to control the presence andthe concentration of S. aureus in foodso Use of phages as food additivesStarter culture phagesSpecific to the strains of thestarter cultureSmall lytic spectrumResponsible for the rejection ofbatches of milk and cheeseS. aureus phagesSpecific to staphylococciPolyvalentCould reduce the number ofbatch rejections (?)5


o Use of phages as food additives:oNo change on organoleptic properties offoodsooListex P100, recognized as safe fortheir use to control Listeriamonocytogenes (FDA, 2007)AgriPhage-CMM anti-Clavibactermichiganensis (Health Canada, 2012)http://www.listex-p100.com/6


o Conditions to use phages as food additives:o Virulent phageo Absence of virulence factors (e.g., toxins)o Large host rangeo Efficiency in the food matrixo Phage cocktail7


Three families of Caudovirales, dsDNAMyoviridaeSiphoviridae3 morphotypesPodoviridae2 morphotypesPyophage, Team1,Team2, phi812, K3A, M SA , LH1, LH1-MUTP68, 44AHJD8


Phages isolated from milk samples are moreadapted in inhibiting S. aureus strains in cheesesThe activity of staphylococcal phages occursduring cheese production and maturation9


1. Selection and identification of S. aureusstrains isolated from raw milk samples2. Collection, isolation, and characterization ofS. aureus phages3. Testing of phage usage during two steps ofCheddar cheese production4. Testing of cocktail efficiency against S. aureusduring Cheddar cheese production10


ooFourty strains of S. aureus isolated from mastitissent by CBMRNooAll Canadian provincesDifferent types of sampleso Mastitis scores from 0 to 3One S. aureus strain isolated from Quebec rawmilk12


Three families of Caudovirales, dsDNAMyoviridaeSiphoviridae3 morphotypesPodoviridae2 morphotypesPyophage, Team1,Team2, phi812, K3A, M SA , LH1, LH1-MUTP68, 44AHJD15


Raw milk samples:o Centrifugation5 ml of milk sample5 ml of TSB 2X containingan overnight culture of S.aureus37 ºC16


Phage LH1o Siphoviridae of B2 morphotypeo Infectious phageo Host strain : S. aureus 01o Turbid solution after amplification17


Natural mutant of LH1, LH1-MUTClear lysateEl Haddad, L., and S. Moineau. 2013. Appl. Environ. Microbiol. 79(8):2828-283218


Heat treatment73 ºC for 20 secRipening1 month at4ºC21


oTemperature/time of heat treatment:o73 ºC for 20 secondsoMedium:oUltrafiltrated pasteurized milk with 2% fat contentoPhages:Myoviridae Podoviridae Siphoviridae CocktailsTeam1 P68 LH1-MUT phi812/K/LH1-MUTphi812 44AHJD phi2 phi812/P68/LH1-MUTK3AM SA 22


Reduction of the phage concentration (Log units) after73ºC for 20 secondsSiphoviridaeLH1-MUT1,4 ±0,5phi20,6 ±0,1M SA2,9 ±0,43A4,2 ±0,2MyoviridaeTeam14,0 ±1,0phi8122,6 ±0,5K3,5 ±0,2PodoviridaeP682,7 ±0,144AHJD3,6 ±0,7Cocktailsphi812/K/LH1-MUT2,2 ±0,7phi812/P68/LH1-MUT2,8 ±0,323


Reduction of the phage concentration (Log units) after73ºC for 20 secondsSiphoviridaeLH1-MUT1,4 ±0,5phi20,6 ±0,1M SA2,9 ±0,43A4,2 ±0,2MyoviridaeTeam14,0 ±1,0phi8122,6 ±0,5K3,5 ±0,2PodoviridaeP682,7 ±0,144AHJD3,6 ±0,7Cocktailsphi812/K/LH1-MUT2,2 ±0,7phi812/P68/LH1-MUT2,8 ±0,324


Heat treatment73ºC for 20 seco Phages LH1-MUT andphi2, isolated from rawmilk, were more resistantthan the restRipening1 month at4ºC25


o Normal conditions of curd:o 1.77% of salto 39% of humidityo Using lyophilized cheddaro Reducing the pH to 5.5 with acidified water(Adapted from Lessard et al. 2012. Appl. Environ. Microbiol.)o Phage at a concentration of 10 7 pfu/gMyoviridae Podoviridae Siphoviridae Phage cocktailsTeam1KP6844AHJDLH1-MUT3Aphi2T1/P68/LH1-MUTK/44AHJD/phi2o Sampling time: 0, 4, 7, 14, and 28 days26


+10 gramsAcidifiedwaterPeptonewaterTSA Sodiumcitrate at45ºC (90mL)4ºC95%vacuumed27


Resistance of phages in curd, pH 5.5 at 4ºC(T1-P68-MUT) (K-44AHJD-2)28


Heat treatment73 ºC for 20 seco Phages LH1-MUT andphi2, isolated from rawmilk, were more resistantthan the restRipening1 month at4ºCo All the phages resist tothese conditions29


o In the presence of S. aureus :o SE production in the absence/presence ofphageso Efficiency of staphylococcal phage cocktailsduring Cheddar cheese production30


Starter culture phagesSpecific to the strains of thestarter cultureSmall lytic spectrumResponsible for the rejection ofbatches of milk and cheeseS. aureus phagesSpecific to staphylococciPolyvalentCould reduce the number ofbatch rejections (?)o Polyvalent phageso Beyond the specieso GRAS starter, S. xylosuso Propagation of phages on a non-pathogenicstrain31


o Phage Team1 amplified on S. aureus/ S. xylosus:o No genetic difference on the sequenced andanalyzed 140Kb32


o Implementation of a technique for the isolation of new S.aureus phages from raw milk sampleso More resistant to heat treatment of milko Use of S. aureus strains isolated from the same mediumo New strategy for the selection of polyvalent and resistantphageso Phages amplified on S. aureus and S. xylosus aregenetically identical33


o Hypothesiso Objectiveso Selectiono Parameterso Types of milko Initial inoculationo Agitationo The time to adding the phageo Cheddar cheeseo Conclusiono Perspectives


The activity of anti-staphylococcal phagesdepends on various parameters related tothe type of milk and the process of cheesemaking


1. Selection of an active phage in raw milkfrom a collection of phages2. Determination of parameters thatimprove the lytic action of phage in milk3. Phage activity was followed during amini Cheddar cheese manufacturing


%Moderately contaminated milkTotal staphylococci= 10 2 UFC/mL350300250200150100KTeam 2Φ812PyophageMost contaminated milkTotal staphylococci= 10 4 UFC/mL50Team 100 5 10 15 20 25time (h)Assess of phage titers in raw milk


%10090807060504030201000 1 2 3 4 5time (h)KTeam 2Φ812PyophageTeam 1Assess of phage titers during the time


Steps of the Pearce test(Pearce L.E., 1969. J. Dairy Technol.)


o Coagulase-negative staphylococci (CNS)o Starter for the manufacture of sausageso Microflora surface for dried meats and cheeses(Munster)


Follow-up of phage titers of phage K in differenttypes of milk


Growth of S. xylosus in TSB with different initial contamination in thepresence of phage K• CI3 = 10 3 CFU/mL• CI5 = 10 5 CFU/mL• CI6 = 10 6 CFU/mL• Phage K (MOI = 100)


Growth of S. xylosus in TSB in the presence of phage K• Initial inoculation = 10 5 CFU/mL• Phage K (MOI = 100)


Growth of S. xylosus in TSB in the presence of phage K• Initial inoculation =10 6 CFU/mL• Phage K (MOI = 10)


o Pasteurized milko Initial contamination= 10 5 ,10 6 CFU/mLo Phage addition: at the beginning ofmanufacturingo Keep a slight continuous agitation


Population of S. xylosus and phage K (%) during the manufacture ofCheddarS. xylosusPhage KMilk Curd Whey


o The lytic activity of anti-staphylococcal phages is optimal inpasteurized milko The activity depends on the initial contamination ofstaphylococci (minimum contamination of 10 5 CFU/mL)o Continuous slight stirring is needed to promote contactbetween the phage and its host cello During cheese production phages seem to be retained inthe curdo In a solid matrix their action appears to be inhibit


o Using a cocktail of phageso Cheddar cheese production from milk contaminatedwith a strain of S. aureus in the presence of acocktail of phageso Effectiveness of phages in other types of cheeseproduction


o Reduction of food contamination risks causedby S. aureuso Improvement of food safety50


Laboratory of Prof. Sylvain MoineauLaboratory of Dr Daniel St-GelaisoooProf. Steve LabrieDr Claude ChampagneFundings : AAC, MAPAQ,Novalait et FRQNT

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