Bacteriophages or, simply, phages (bacteria-infecting viruses) are ubiquitous. They are now acknowledged as the most predominant biological entities on our planet. Up to 108 phages can be found in a single drop of sea water [1]. Scientists have also long tried to use phages (or now phage-derived proteins) to treat diseases such as dysentery or staphylococcal infections [2]. Phages are obligate parasites and most phage multiplication cycle end with cell lysis and the release of hundreds of new virions ready to infect neighbouring cells (for a review on phage biology see [3]). One of the key roles of phages is to balance the bacterial population in every shared environment thereby challenging bacteria to rapidly evolve. Phages can also sometimes turn an industrial microbiologist’s professional life into a nightmare! A biotechnology process that relies on the use of bacteria to produce a molecule or make a product can be disrupted by phages. Problems due to the presence of phages were reported in the food, chemical, pharmaceutical, feed and pesticide industries [4]. However, the dairy industry is probably the one in which phage problems are the most documented.
The manufacture of cheese requires the inoculation of 107 carefully selected bacterial cells (known as starter cultures) per ml of pasteurized milk to control the fermentation and to obtain high-quality end-products. Starter cultures are a combination of various lactic acid bacteria (LAB), usually strains of Lactococcus lactis, Streptococcus thermophilus, Leuconostoc sp., and/or Lactobacillus sp. Considering that 1014 bacterial cells are needed to produce 1 ton of cheese, it is clear that LAB are of considerable interest to the cheese industry. In the non-sterile environment of raw or heat-treated milk, the added LAB cells will come into contact with virulent phages found in milk [4]. Although phage concentration is usually low in milk, a specific phage population can increase rapidly if phage-sensitive cells are present in the starter culture. The consequent lysis of a large number of sensitive cells will delay or even halt the milk fermentation process leading to low-quality products. In worse cases, the inoculated milk must be discarded. For decades, the dairy industry has been dealing with this natural phenomenon and has relied on an array of control measures, notably adapted factory design, improved sanitation, process changes, specific culture medium, strain rotation, and the use of phage-resistant strains.
The first description of phages affecting a dairy starter culture was reported by Whitehead and Cox in 1935 and since then, the field has seen significant improvements, particularly in the areas of phage genetics, ecology, and resistance to environmental factors [5]. In fact LAB phages are now among the most studied phages. Nevertheless, phage contamination can still occur nowadays leading to product variability and to reduce productivity [6,7]. Phages can also cause problems in the fast growing probiotic field, where the genotype of the strain is highly valuable [8,9].
The topic of “LAB phages” has been extensively covered in the past 25 years. Many excellent reviews have tackled LAB phages from various perspectives and readers interested to learn more about this topic will find a list of reviews in additional file 1. Hence, this review will mainly focus on recent scientific advances in the LAB phage research field, with a particular focus on the practical/applied aspects related to the dairy fermentation industry. Topics for which we believe additional research is needed will also be highlighted.
Go to:
Sources of contamination
Phages can come from various sources. It is of prime importance to know the potential sources of phages to limit their entry within the manufacturing facilities, which could be deleterious to the fermentation process.
Raw ingredients
Any raw natural ingredient that enters a fermentation facility may contain phages, albeit at low levels. For example, raw milk, which is an ecological niche for some LAB, is well known to contain phages [6]. Because milk is collected from different farms, phage biodiversity is amplified within milk silos. Since phages can easily propagate in a liquid medium such as milk and since they can also diffuse in gel-like media, only a few sensitive cells are needed to rapidly increase phage levels in a given environment [10]. Using a multiplex PCR method, lactococcal and streptococcal phages have been detected in 37% of the milk samples used for yogurt production in Spain [11], while microbiological approaches demonstrated that 9% of milk samples from various geographical areas in Spain contained L. lactis phage [12]. These numbers can be higher in whey samples or final products since phages can propagate during most fermentation processes [12,13]. Titers as high as 109 PFU per ml of cheese whey have been reported [14].
Depending on the frequencies of phage attacks and the size of the facilities, it may be advisable to analyse milk (or other ingredients) for the presence of phages before beginning the fermentation process to confirm that the initial phage load does not represent a significant risk of fermentation failure. If the ingredients are thought to pose a risk, they can be treated to reduce phage levels or used for other processes that will not be affected by phages. Effective cleaning procedures must be also in-place to reduce to the initial phage load.
Processed or recycled ingredients
The milk fermentation industry may reuse whey proteins to improve the taste or texture of a final product, to increase its nutrient value [15-17], to standardize milk before the fermentation process or to increase the yield [4,18]. Upon whey or milk protein concentration, phages may remain in the whey protein concentrate (liquid or dried) and contaminate the products to which it is added [19]. When using membranes to separate whey components, it is highly possible that phages will be retained by ultrafiltration and/or microfiltration [20]. Depending on whether the retentate or the permeate is used, phages might still be present and cause problems in subsequent transformation processes. Ideally, milk by-products should either be treated to inactivate the phages or be used in a type of fermentation that is driven by different starter cultures. For example, if the whey was collected from a cheddar fermentation made using mesophilic starter cultures, by-products of this whey could safely be used in yogurt manufacture or in a cheese process requiring thermophilic cultures. In addition, the use of concentrated milk products from another dairy plant (which may use different starter cultures) can offer additional protection. Although the latter will also most likely increase the phage biodiversity within the factory.
Phage reservoir
One perceived source of phages is the starter culture itself. When a temperate phage enters a strain, it can either start the lytic cycle or its genome can integrate into the bacterial chromosome and follow bacterial multiplication. When bacteria carry such a prophage, the cell is called a lysogen. Different bacterial stresses such as heat, salts, antimicrobials, starvation or UV can induce the prophage and trigger the lytic cycle [21,22]. Thus, the use of lysogenic strains in a starter culture may lead to cell lysis during fermentation. Induction can also occur naturally and can reach a frequency of up to 9% [23]. Prophages are carried by many LAB strains [24,25] and often more than one prophage is found in a genome. The most recent analysis revealed that 25 out of 30 commercial, collection or dairy-isolated Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus were found to carry inducible prophages [26]. It should be noted that most starter culture suppliers will test their strains for the presence of prophages and their natural induction rate. Usually, lysogenic strains carrying easily inducible prophages will not find their way into commercial products. Of note, phage induction assays cannot be readily performed with undefined starter culture as the exact strain composition of this type of starter is unknown.
The presence of prophage in a strain used as part of a starter culture may not always negatively impact the fermentation process [27]. It has been alleged that prophages have beneficial impact on the organoleptic properties of cheeses through the expression of prophage-encoded endolysins that could stimulate autolysis and the release of intracellular flavor generating-enzymes [28]. Prophage genes also have the advantage of protecting cells against superinfection by other phages, as it was observed in lactococci and S. thermophilus[29-32].
Prophages can also act as a reservoir of viral genes and participate in the recombination and release of new virulent phages with enhanced host range and new peculiarities [25,33]. For example, propagation of a virulent phage on a strain carrying a phage resistant mechanism led to the emergence of phage mutants insensitive to the anti-phage system. Comparative analysis of the genomes of the phage mutants revealed that the wild-type phage extensively evolved by large-scale homologous and non-homologous recombinations with the inducible prophage present in the host strain. Thus, natural phage defence mechanisms and prophage elements are contributing to the evolution of the virulent phage population [33]. Another example is the recent isolation of virulent phage infecting a probiotic L. paracasei strain which has a similar host range as a mitomycin C-induced phage from another L. paracasei strain [26,34].
Air/surfaces
Whereas phage contamination of milk is probably the most obvious and primary source of phages in a dairy plant, dissemination routes of contaminants can be more complicated to identify. Recently, the presence of airborne lactococcal phages in a cheese plant was investigated because it had been ra
Bacteriophages or, simply, phages (bacteria-infecting viruses) are ubiquitous. They are now acknowledged as the most predominant biological entities on our planet. Up to 108 phages can be found in a single drop of sea water [1]. Scientists have also long tried to use phages (or now phage-derived proteins) to treat diseases such as dysentery or staphylococcal infections [2]. Phages are obligate parasites and most phage multiplication cycle end with cell lysis and the release of hundreds of new virions ready to infect neighbouring cells (for a review on phage biology see [3]). One of the key roles of phages is to balance the bacterial population in every shared environment thereby challenging bacteria to rapidly evolve. Phages can also sometimes turn an industrial microbiologist’s professional life into a nightmare! A biotechnology process that relies on the use of bacteria to produce a molecule or make a product can be disrupted by phages. Problems due to the presence of phages were reported in the food, chemical, pharmaceutical, feed and pesticide industries [4]. However, the dairy industry is probably the one in which phage problems are the most documented.
The manufacture of cheese requires the inoculation of 107 carefully selected bacterial cells (known as starter cultures) per ml of pasteurized milk to control the fermentation and to obtain high-quality end-products. Starter cultures are a combination of various lactic acid bacteria (LAB), usually strains of Lactococcus lactis, Streptococcus thermophilus, Leuconostoc sp., and/or Lactobacillus sp. Considering that 1014 bacterial cells are needed to produce 1 ton of cheese, it is clear that LAB are of considerable interest to the cheese industry. In the non-sterile environment of raw or heat-treated milk, the added LAB cells will come into contact with virulent phages found in milk [4]. Although phage concentration is usually low in milk, a specific phage population can increase rapidly if phage-sensitive cells are present in the starter culture. The consequent lysis of a large number of sensitive cells will delay or even halt the milk fermentation process leading to low-quality products. In worse cases, the inoculated milk must be discarded. For decades, the dairy industry has been dealing with this natural phenomenon and has relied on an array of control measures, notably adapted factory design, improved sanitation, process changes, specific culture medium, strain rotation, and the use of phage-resistant strains.
The first description of phages affecting a dairy starter culture was reported by Whitehead and Cox in 1935 and since then, the field has seen significant improvements, particularly in the areas of phage genetics, ecology, and resistance to environmental factors [5]. In fact LAB phages are now among the most studied phages. Nevertheless, phage contamination can still occur nowadays leading to product variability and to reduce productivity [6,7]. Phages can also cause problems in the fast growing probiotic field, where the genotype of the strain is highly valuable [8,9].
The topic of “LAB phages” has been extensively covered in the past 25 years. Many excellent reviews have tackled LAB phages from various perspectives and readers interested to learn more about this topic will find a list of reviews in additional file 1. Hence, this review will mainly focus on recent scientific advances in the LAB phage research field, with a particular focus on the practical/applied aspects related to the dairy fermentation industry. Topics for which we believe additional research is needed will also be highlighted.
Go to:
Sources of contamination
Phages can come from various sources. It is of prime importance to know the potential sources of phages to limit their entry within the manufacturing facilities, which could be deleterious to the fermentation process.
Raw ingredients
Any raw natural ingredient that enters a fermentation facility may contain phages, albeit at low levels. For example, raw milk, which is an ecological niche for some LAB, is well known to contain phages [6]. Because milk is collected from different farms, phage biodiversity is amplified within milk silos. Since phages can easily propagate in a liquid medium such as milk and since they can also diffuse in gel-like media, only a few sensitive cells are needed to rapidly increase phage levels in a given environment [10]. Using a multiplex PCR method, lactococcal and streptococcal phages have been detected in 37% of the milk samples used for yogurt production in Spain [11], while microbiological approaches demonstrated that 9% of milk samples from various geographical areas in Spain contained L. lactis phage [12]. These numbers can be higher in whey samples or final products since phages can propagate during most fermentation processes [12,13]. Titers as high as 109 PFU per ml of cheese whey have been reported [14].
Depending on the frequencies of phage attacks and the size of the facilities, it may be advisable to analyse milk (or other ingredients) for the presence of phages before beginning the fermentation process to confirm that the initial phage load does not represent a significant risk of fermentation failure. If the ingredients are thought to pose a risk, they can be treated to reduce phage levels or used for other processes that will not be affected by phages. Effective cleaning procedures must be also in-place to reduce to the initial phage load.
Processed or recycled ingredients
The milk fermentation industry may reuse whey proteins to improve the taste or texture of a final product, to increase its nutrient value [15-17], to standardize milk before the fermentation process or to increase the yield [4,18]. Upon whey or milk protein concentration, phages may remain in the whey protein concentrate (liquid or dried) and contaminate the products to which it is added [19]. When using membranes to separate whey components, it is highly possible that phages will be retained by ultrafiltration and/or microfiltration [20]. Depending on whether the retentate or the permeate is used, phages might still be present and cause problems in subsequent transformation processes. Ideally, milk by-products should either be treated to inactivate the phages or be used in a type of fermentation that is driven by different starter cultures. For example, if the whey was collected from a cheddar fermentation made using mesophilic starter cultures, by-products of this whey could safely be used in yogurt manufacture or in a cheese process requiring thermophilic cultures. In addition, the use of concentrated milk products from another dairy plant (which may use different starter cultures) can offer additional protection. Although the latter will also most likely increase the phage biodiversity within the factory.
Phage reservoir
One perceived source of phages is the starter culture itself. When a temperate phage enters a strain, it can either start the lytic cycle or its genome can integrate into the bacterial chromosome and follow bacterial multiplication. When bacteria carry such a prophage, the cell is called a lysogen. Different bacterial stresses such as heat, salts, antimicrobials, starvation or UV can induce the prophage and trigger the lytic cycle [21,22]. Thus, the use of lysogenic strains in a starter culture may lead to cell lysis during fermentation. Induction can also occur naturally and can reach a frequency of up to 9% [23]. Prophages are carried by many LAB strains [24,25] and often more than one prophage is found in a genome. The most recent analysis revealed that 25 out of 30 commercial, collection or dairy-isolated Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus were found to carry inducible prophages [26]. It should be noted that most starter culture suppliers will test their strains for the presence of prophages and their natural induction rate. Usually, lysogenic strains carrying easily inducible prophages will not find their way into commercial products. Of note, phage induction assays cannot be readily performed with undefined starter culture as the exact strain composition of this type of starter is unknown.
The presence of prophage in a strain used as part of a starter culture may not always negatively impact the fermentation process [27]. It has been alleged that prophages have beneficial impact on the organoleptic properties of cheeses through the expression of prophage-encoded endolysins that could stimulate autolysis and the release of intracellular flavor generating-enzymes [28]. Prophage genes also have the advantage of protecting cells against superinfection by other phages, as it was observed in lactococci and S. thermophilus[29-32].
Prophages can also act as a reservoir of viral genes and participate in the recombination and release of new virulent phages with enhanced host range and new peculiarities [25,33]. For example, propagation of a virulent phage on a strain carrying a phage resistant mechanism led to the emergence of phage mutants insensitive to the anti-phage system. Comparative analysis of the genomes of the phage mutants revealed that the wild-type phage extensively evolved by large-scale homologous and non-homologous recombinations with the inducible prophage present in the host strain. Thus, natural phage defence mechanisms and prophage elements are contributing to the evolution of the virulent phage population [33]. Another example is the recent isolation of virulent phage infecting a probiotic L. paracasei strain which has a similar host range as a mitomycin C-induced phage from another L. paracasei strain [26,34].
Air/surfaces
Whereas phage contamination of milk is probably the most obvious and primary source of phages in a dairy plant, dissemination routes of contaminants can be more complicated to identify. Recently, the presence of airborne lactococcal phages in a cheese plant was investigated because it had been ra
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