Direct Quantitative Detection and Identification of Lactococcal Bacteriophages from Milk and Whey by Real-Time PCR: Application for the Detection of Lactococcal Bacteriophages in Goat's Raw Milk Whey in France
The presence of Lactococcus bacteriophages in milk can partly or completely inhibit milk fermentation. To prevent the problems associated with the bacteriophages, the real-time PCR was developed in this study for direct detection from whey and milk of three main groups of Lactococcus bacteriophages, c2, 936, and P335. The optimization of DNA extraction protocol from complex matrices such as whey and milk was optimized allowed the amplification of PCR without any matrix and nontarget contaminant interference. The real-time PCR program was specific and with the detection limit of 102 PFU/mL. The curve slopes were ？3.49, ？3.69, and ？3.45 with the amplification efficiency estimated at 94%, 94%, and 98% and the correlation coefficient ( ) of 0.999, 0.999, and 0.998 for c2, 936 and P335 group, respectively. This method was then used to detect the bacteriophages in whey and goat's raw milk coming from three farms located in the Rh？ne-Alpes region (France). 1. Introduction Raw milk is a complex microbial ecosystem containing different microorganisms, spoiling bacteria, pathogens, and technological flora such as lactic acid bacteria. The last bacteria participate in the cheese-making process and ripening [1]. Raw milk may also contain the bacteriophages of lactic acid bacteria which could present a risk during dairy processing. The lactic acid bacteria may be infected by the bacteriophages leading to a slower acidification process or the end of it [2]. The disappearance of several flora may modify ripening processes. The contamination of bacteriophages can result from the environment, raw milk, machines, and airborne bacteria. Moreover, some strains of bacteriophages are resistant to pasteurization [3]. The whey separated after the first fermentation can be used for the next fermentation in cheese making, (backslopping). This practice allows the reproduction of a large number of microbial flora technologically [4]. But this practice may reproduce also the contamination of bacteriophages which are suspected to be a cause of acidification incidents, especially in cheese processing using raw milk [1, 3]. It seemed essential to determine the biodiversity of bacteriophages in raw milk and whey to understand the incidents related to acidification. The diversity of bacteriophages in most important dairy strains (Streptococcus thermophilus, Lactobacillus, and Lactococcus) was studied in several countries: in Canada [5], in Spain [6], in Argentina [7], and in Belarus [8]. In these studies, lactococcal phages were mostly studied for Lactococcus lactis application in many
References
[1]
E. Franciosi, L. Settanni, A. Cavazza, and E. Poznanski, “Biodiversity and technological potential of wild lactic acid bacteria from raw cows' milk,” International Dairy Journal, vol. 19, no. 1, pp. 3–11, 2009.
[2]
G. M. Rousseau and S. Moineau, “Evolution of Lactococcus lactis phages within a cheese factory,” Applied and Environmental Microbiology, vol. 75, no. 16, pp. 5336–5344, 2009.
[3]
C. Madera, C. Monjardín, and J. E. Suárez, “Milk contamination and resistance to processing conditions determine the fate of Lactococcus lactis bacteriophages in dairies,” Applied and Environmental Microbiology, vol. 70, no. 12, pp. 7365–7371, 2004.
[4]
M. Dalmasso, D. Hennequin, C. Duc, and Y. Demarigny, “Influence of backslopping on the acidifications curves of “Tomme” type cheeses made during 10 successive days,” Journal of Food Engineering, vol. 92, no. 1, pp. 50–55, 2009.
[5]
H. Deveau, S. J. Labrie, M. C. Chopin, and S. Moineau, “Biodiversity and classification of lactococcal phages,” Applied and Environmental Microbiology, vol. 72, no. 6, pp. 4338–4346, 2006.
[6]
B. del Rio, A. G. Binetti, M. C. Martín, M. Fernández, A. H. Magadán, and M. A. Alvarez, “Multiplex PCR for the detection and identification of dairy bacteriophages in milk,” Food Microbiology, vol. 24, no. 1, pp. 75–81, 2007.
[7]
A. Quiberoni, D. Tremblay, H. W. Ackermann, S. Moineau, and J. A. Reinheimer, “Diversity of Streptococcus thermophilus phages in a large-production cheese factory in Argentina,” Journal of Dairy Science, vol. 89, no. 10, pp. 3791–3799, 2006.
[8]
D. Lillehaug, “An improved plaque assay for poor plaque-producing temperate lactococcal bacteriophages,” Journal of Applied Microbiology, vol. 83, no. 1, pp. 85–90, 1997.
[9]
O. Michelsen, A. Cuesta-Dominguez, B. Albrechtsen, and P. R. Jensen, “Detection of bacteriophage-infected cells of Lactococcus lactis by using flow cytometry,” Applied and Environmental Microbiology, vol. 73, no. 23, pp. 7575–7581, 2007.
[10]
A. Raiski and N. Belyasova, “Biodiversity of Lactococcus lactis bacteriophages in the Republic of Belarus,” International Journal of Food Microbiology, vol. 130, no. 1, pp. 1–5, 2009.
[11]
M. C. Martín, B. del Rio, N. Martínez, A. H. Magadán, and M. A. Alvarez, “Fast real-time polymerase chain reaction for quantitative detection of Lactobacillus delbrueckii bacteriophages in milk,” Food Microbiology, vol. 25, no. 8, pp. 978–982, 2008.
[12]
M. Kubista, J. M. Andrade, M. Bengtsson et al., “The real-time polymerase chain reaction,” Molecular Aspects of Medicine, vol. 27, no. 2-3, pp. 95–125, 2006.
[13]
A. G. Binetti, B. Del Río, M. Cruz Martín, and M. A. álvarez, “Detection and characterization of Streptococcus thermophilus bacteriophages by use of the antireceptor gene sequence,” Applied and Environmental Microbiology, vol. 71, no. 10, pp. 6096–6103, 2005.
[14]
S. Labrie and S. Moineau, “Multiplex PCR for detection and identification of lactococcal bacteriophages,” Applied and Environmental Microbiology, vol. 66, no. 3, pp. 987–994, 2000.
[15]
D. Verreault, L. Gendron, G. M. Rousseau, et al., “Detection of airborne lactococcal bacteriophages in cheese manufacturing plants,” Applied and Environmental Microbiology, vol. 77, no. 2, pp. 491–497, 2011.
[16]
P. Martorell, A. Querol, and M. T. Fernández-Espinar, “Rapid identification and enumeration of Saccharomyces cerevisiae cells in wine by real-time PCR,” Applied and Environmental Microbiology, vol. 71, no. 11, pp. 6823–6830, 2005.
[17]
G. Amagliani, C. Giammarini, E. Omiccioli, G. Brandi, and M. Magnani, “Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods,” Food Control, vol. 18, no. 9, pp. 1137–1142, 2007.
[18]
A. K. Szczepańska, M. S. Hejnowicz, P. Ko？akowski, and J. Bardowski, “Biodiversity of Lactococcus lactis bacteriophages in Polish dairy environment,” Acta Biochimica Polonica, vol. 54, no. 1, pp. 151–158, 2007.
[19]
S. Labrie and S. Moineau, “Complete genomic sequence of bacteriophage ul36: demonstration of phage heterogeneity within the P335 quasi-species of lactococcal phages,” Virology, vol. 296, no. 2, pp. 308–320, 2002.
[20]
J. D. Bouchard and S. Moineau, “Homologous recombination between a lactococcal bacteriophage and the chromosome of its host strain,” Virology, vol. 270, no. 1, pp. 65–75, 2000.
