Personne :
Labrie, Steve

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Labrie
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Steve
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Université Laval. Faculté des sciences de l'agriculture et de l'alimentation
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  • Publication
    Restreint
    Complete genomic sequence of bacteriophage ul36: demonstration of phage heterogeneity within the P335 quasi-species of lactococcal phages
    (Elsevier, 2002-05-10) Labrie, Steve; Moineau, Sylvain
    The complete genomic sequence of the Lactococcus lactis virulent phage ul36 belonging to P335 lactococcal phage species was determined and analyzed. The genomic sequence of this lactococcal phage contained 36,798 bp with an overall G+C content of 35.8 mol %. Fifty-nine open reading frames (ORFs) of more than 40 codons were found. N-terminal sequencing of phage structural proteins as well as bioinformatic analysis led to the attribution of a function to 24 ORFs (41%). A lysogeny module was found within the genome of this virulent phage. The putative integrase gene seems to be the product of a horizontal transfer because it is more closely related to Streptococcus pyogenes phages than it is to L. lactis phages. Comparative genome analysis with six complete genomes of temperate P335-like phages confirmed the heterogeneity among phages of P335 species. A dUTPase gene is the only conserved gene among all P335 phages analyzed as well as the phage BK5-T. A genetic relationship between P335 phages and the phage-type of the BK5-T species was established. Thus, we proposed that phage BK5-T be included within the P335 species and thereby reducing the number of lactococcal phage species to 11.
  • Publication
    Accès libre
    Metatranscriptome analysis of fungal strains Penicillium and Geotrichum reveal cheese matrix breakdown and potential development of sensory properties of ripened Camembert-type cheese
    (MEDLINE/PubMed (U.S. National Library of Medicine), 2014-03-26) Viel, Catherine; Lessard, Marie-Hélène; Boyle, Brian; Labrie, Steve; St-Gelais, Daniel
    Camembert-type cheese ripening is driven mainly by fungal microflora including Geotrichum candidum and Penicillium camemberti. These species are major contributors to the texture and flavour of typical bloomy rind cheeses. Biochemical studies showed that G. candidum reduces bitterness, enhances sulphur flavors through amino acid catabolism and has an impact on rind texture, firmness and thickness, while P. camemberti is responsible for the white and bloomy aspect of the rind, and produces enzymes involved in proteolysis and lipolysis activities. However, very little is known about the genetic determinants that code for these activities and their expression profile over time during the ripening process.
  • Publication
    Accès libre
    The composition of Camembert cheese-ripening cultures modulates both mycelial growth and appearance
    (American Society of Microbiology, 2012-01-13) Lessard, Marie-Hélène; Bélanger, Gaétan; Labrie, Steve; St-Gelais, Daniel
    The fungal microbiota of bloomy-rind cheeses, such as Camembert, forms a complex ecosystem that has not been well studied, and its monitoring during the ripening period remains a challenge. One limitation of enumerating yeasts and molds on traditional agar media is that hyphae are multicellular structures, and colonies on a petri dish rarely develop from single cells. In addition, fungi tend to rapidly invade agar surfaces, covering small yeast colonies and resulting in an underestimation of their number. In this study, we developed a real-time quantitative PCR (qPCR) method using TaqMan probes to quantify a mixed fungal community containing the most common dairy yeasts and molds: Penicillium camemberti, Geotrichum candidum, Debaryomyces hansenii, and Kluyveromyces lactis on soft-cheese model curds (SCMC). The qPCR method was optimized and validated on pure cultures and used to evaluate the growth dynamics of a ripening culture containing P. camemberti, G. candidum, and K. lactis on the surface of the SCMC during a 31-day ripening period. The results showed that P. camemberti and G. candidum quickly dominated the ecosystem, while K. lactis remained less abundant. When added to this ecosystem, D. hansenii completely inhibited the growth of K. lactis in addition to reducing the growth of the other fungi. This result was confirmed by the decrease in the mycelium biomass on SCMC. This study compares culture-dependent and qPCR methods to successfully quantify complex fungal microbiota on a model curd simulating Camembert-type cheese.
  • Publication
    Restreint
    Efficacy of two Staphylococcus aureus phage cocktails in cheese production
    (Elsevier Science Publishers, 2015-10-05) Labrie, Steve; Roy, Jean-Pierre; Moineau, Sylvain; Khalil, Georges E.; Champagne, Claude P.; El Haddad, Lynn; St-Gelais, Daniel
    Staphylococcus aureus is one of the most prevalent pathogenic bacteria contaminating dairy products. In an effort to reduce food safety risks, virulent phages are investigated as antibacterial agents to control foodborne pathogens. The aim of this study was to compare sets of virulent phages, design phage cocktails, and use them in a cocktail to control pathogenic staphylococci in cheese. Six selected phages belonging to the three Caudovirales families (Myoviridae, Siphoviridae, Podoviridae) were strictly lytic, had a broad host range, and did not carry genes coding for virulence traits in their genomes. However, they were sensitive to pasteurization. At MOI levels of 15, 45, and 150, two anti-S. aureus phage cocktails, each containing three phages, one from each of the three phage families, eradicated a 106 CFU/g S. aureus population after 14 days of Cheddar cheese curd ripening at 4 °C. The use of these phages did not trigger over-production of S. aureus enterotoxin C. The use of phage cocktails and their rotation may prevent the emergence of phage resistant bacterial strains.
  • Publication
    Restreint
    Multiplex PCR for detection and identification of lactococcal bacteriophages
    (American Society for Microbiology, 2000-03-01) Labrie, Steve; Moineau, Sylvain
    Three genetically distinct groups of Lactococcus lactis phages are encountered in dairy plants worldwide, namely, the 936, c2, and P335 species. The multiplex PCR method was adapted to detect, in a single reaction, the presence of these species in whey samples or in phage lysates. Three sets of primers, one for each species, were designed based on conserved regions of their genomes. The c2-specific primers were constructed using the major capsid protein gene (mcp) as the target. The mcp sequences for three phages (eb1, Q38, and Q44) were determined and compared with the two available in the databases, those for phages c2 and bIL67. An 86.4% identity was found over the five mcp genes. The gene of the only major structural protein (msp) was selected as a target for the detection of 936-related phages. The msp sequences for three phages (p2, Q7, and Q11) were also established and matched with the available data on phages sk1, bIL170, and F4-1. The comparison of the six msp genes revealed an 82.2% identity. A high genomic diversity was observed among structural proteins of the P335-like phages suggesting that the classification of lactococcal phages within this species should be revised. Nevertheless, we have identified a common genomic region in 10 P335-like phages isolated from six countries. This region corresponded to orfF17-orf18 of phage r1t and orf20-orf21 of Tuc2009 and was sequenced for three additional P335 phages (Q30, P270, and ul40). An identity of 93.4% within a 739-bp region of the five phages was found. The detection limit of the multiplex PCR method in whey was 104 to 107 PFU/ml and was 103 to 105 PFU/ml with an additional phage concentration step. The method can also be used to detect phage DNA in whey powders and may also detect prophage or defective phage in the bacterial genome.
  • Publication
    Accès libre
    Phages of lactic acid bacteria : from genomics to industrial applications
    (American Society for Microbiology, 2002-08-01) Tremblay, Denise; Labrie, Steve; Moineau, Sylvain
    Bacteriophage research has received considerable attention in recent years due to an outburst of studies and editorials about the potential use of phages in treating bacterial infections (see ASM News, September 2001). This research work is fueled by the pressing need to find alternative strategies to antibiotics. Conversely, dairy microbiologists have been trying intensively to get rid of bacteriophages for more than seventy years!
  • Publication
    Restreint
    Multiplex PCR for detection and identification of lactococcal bacteriophages
    (American Society for Microbiology, 2000-03-01) Labrie, Steve; Moineau, Sylvain
    Three genetically distinct groups of Lactococcus lactis phages are encountered in dairy plants worldwide, namely, the 936, c2, and P335 species. The multiplex PCR method was adapted to detect, in a single reaction, the presence of these species in whey samples or in phage lysates. Three sets of primers, one for each species, were designed based on conserved regions of their genomes. The c2-specific primers were constructed using the major capsid protein gene (mcp) as the target. The mcp sequences for three phages (eb1, Q38, and Q44) were determined and compared with the two available in the databases, those for phages c2 and bIL67. An 86.4% identity was found over the five mcp genes. The gene of the only major structural protein (msp) was selected as a target for the detection of 936-related phages. The msp sequences for three phages (p2, Q7, and Q11) were also established and matched with the available data on phages sk1, bIL170, and F4-1. The comparison of the six msp genes revealed an 82.2% identity. A high genomic diversity was observed among structural proteins of the P335-like phages suggesting that the classification of lactococcal phages within this species should be revised. Nevertheless, we have identified a common genomic region in 10 P335-like phages isolated from six countries. This region corresponded to orfF17-orf18 of phage r1t and orf20-orf21 of Tuc2009 and was sequenced for three additional P335 phages (Q30, P270, and ul40). An identity of 93.4% within a 739-bp region of the five phages was found. The detection limit of the multiplex PCR method in whey was 104 to 107 PFU/ml and was 103 to 105 PFU/ml with an additional phage concentration step. The method can also be used to detect phage DNA in whey powders and may also detect prophage or defective phage in the bacterial genome.
  • Publication
    Accès libre
    Genomic organization and molecular analysis of virulent bacteriophage 2972 infecting an exopolysaccharide-producing Streptococcus thermophilus strain
    (American Society for Microbiology, 2005-07-06) Tremblay, Denise; Labonté, Jessica.; Labrie, Steve; Moineau, Sylvain; Fremaux, Christophe; Duplessis, Martin; Lévesque, Céline
    The Streptococcus thermophilus virulent pac-type phage 2972 was isolated from a yogurt made in France in 1999. It is a representative of several phages that have emerged with the industrial use of the exopolysaccharide-producing S. thermophilus strain RD534. The genome of phage 2972 has 34,704 bp with an overall G+C content of 40.15%, making it the shortest S. thermophilus phage genome analyzed so far. Forty-four open reading frames (ORFs) encoding putative proteins of 40 or more amino acids were identified, and bioinformatic analyses led to the assignment of putative functions to 23 ORFs. Comparative genomic analysis of phage 2972 with the six other sequenced S. thermophilus phage genomes confirmed that the replication module is conserved and that cos- and pac-type phages have distinct structural and packaging genes. Two group I introns were identified in the genome of 2972. They interrupted the genes coding for the putative endolysin and the terminase large subunit. Phage mRNA splicing was demonstrated for both introns, and the secondary structures were predicted. Eight structural proteins were also identified by N-terminal sequencing and/or matrix-assisted laser desorption ionization—time-of-flight mass spectrometry. Detailed analysis of the putative minor tail proteins ORF19 and ORF21 as well as the putative receptor-binding protein ORF20 showed the following interesting features: (i) ORF19 is a hybrid protein, because it displays significant identity with both pac- and cos-type phages; (ii) ORF20 is unique; and (iii) a protein similar to ORF21 of 2972 was also found in the structure of the cos-type phage DT1, indicating that this structural protein is present in both S. thermophilus phage groups. The implications of these findings for phage classification are discussed.
  • Publication
    Restreint
    Receptor-binding protein of lactococcus lactis phages : identification and characterization of the saccharide receptor-binding site
    (American Society for Microbiology, 2006-03-17) Tremblay, Denise; Tegoni, Mariella; Labrie, Steve; Spinelli, Silvia; Moineau, Sylvain; Campanacci, Valérie; Blangy, Stéphanie; Huyghe, Céline; Desmyter, Aline; Cambillau, Christian
    Phage p2, a member of the lactococcal 936 phage species, infects Lactococcus lactis strains by binding initially to specific carbohydrate receptors using its receptor-binding protein (RBP). The structures of p2 RBP, a homotrimeric protein composed of three domains, and of its complex with a neutralizing llama VH domain (VHH5) have been determined (S. Spinelli, A. Desmyter, C. T. Verrips, H. J. de Haard, S. Moineau, and C. Cambillau, Nat. Struct. Mol. Biol. 13:85–89, 2006). Here, we show that VHH5 was able to neutralize 12 of 50 lactococcal phages belonging to the 936 species. Moreover, escape phage mutants no longer neutralized by VHH5 were isolated from 11 of these phages. All of the mutations (but one) cluster in the RBP/VHH5 interaction surface that delineates the receptor-binding area. A glycerol molecule, observed in the 1.7-Å resolution structure of RBP, was found to bind tightly (Kd = 0.26 M) in a crevice located in this area. Other saccharides bind RBP with comparable high affinity. These data prove the saccharidic nature of the bacterial receptor recognized by phage p2 and identify the position of its binding site in the RBP head domain.
  • Publication
    Accès libre
    Improving the safety of Staphylococcus aureus polyvalent phages by their production on a Staphylococcus xylosus strain
    (Public Library of Science, 2014-07-25) Dumaresq, Jeannot; Labrie, Steve; Moineau, Sylvain; Plante, Pier-Luc; Katsarava, Ramaz; El Haddad, Lynn; St-Gelais, Daniel; Ben Abdallah, Nour
    Team1 (vB_SauM_Team1) is a polyvalent staphylococcal phage belonging to the Myoviridae family. Phage Team1 was propagated on a Staphylococcus aureus strain and a non-pathogenic Staphylococcus xylosus strain used in industrial meat fermentation. The two Team1 preparations were compared with respect to their microbiological and genomic properties. The burst sizes, latent periods, and host ranges of the two derivatives were identical as were their genome sequences. Phage Team1 has 140,903 bp of double stranded DNA encoding for 217 open reading frames and 4 tRNAs. Comparative genomic analysis revealed similarities to staphylococcal phages ISP (97%) and G1 (97%). The host range of Team1 was compared to the well-known polyvalent staphylococcal phages phi812 and K using a panel of 57 S. aureus strains collected from various sources. These bacterial strains were found to represent 18 sequence types (MLST) and 14 clonal complexes (eBURST). Altogether, the three phages propagated on S. xylosus lysed 52 out of 57 distinct strains of S. aureus. The identification of phage-insensitive strains underlines the importance of designing phage cocktails with broadly varying and overlapping host ranges. Taken altogether, our study suggests that some staphylococcal phages can be propagated on food-grade bacteria for biocontrol and safety purposes.