Various dairy products and LAB species applied in their production.
The first negative effect of bacteriophages on dairy fermentation was reported in the mid 30s of the XX century . Regardless of sanitary precautions, starter strain rotations and constant development of new phage-resistant bacterial strains, phages remain one of the main and economically most serious sources of fermentation failures. Due to their natural presence in the milk environment, bacteriophages cause problems in industrial dairy fermentations world-wide. Their short latent period, relatively large burst size and/or resistance to pasteurization makes them difficult to eliminate . Phage-induced bacterial cell lysis leads to failed or slow fermentation, decrease in acid production and reduction of milk product quality (e.g. nutritive value, taste, texture, etc.), which in effect cause profound economical losses . An intriguing high number of bacteriophages of
2. Lactic acid bacteria used in dairy industry
Lactic acid bacteria (LAB) comprise different groups of microorganisms, such as
A typical lactococcal mixed starter culture consists of 2-3 well defined strains, which specific properties have significant impact on the texture and flavor of the end product. Nowadays, large dairy plants process up to 106 liters of milk per day, producing annually approximately 107 tons of cheese . Therefore, technological problems in production of cottage and hard cheeses caused by bacteriophage infections have serious economical consequences.
3. Lactic acid bacteria phages – history background, morphology, classification
The history of discovery of bacteriophages originates in the research of Felix d’Herelle and Frederick Twort in the beginning of the XX century and further development of phage biology studies spans the fourth quarter of the last century. Bacteriophages (phages) are defined as viruses that exert their activity against prokaryotic cells – both bacterial as well as archeal.
The name “bacteriophage”derives from the Greek word “phagein”, meaning “to eat”, which points to their destructive action. Bacteriophages exist in two states – extra- and intracellular – which place them half-way between live organisms and non-viable forms. As obligate intracellular parasites their survival is dependent on host organisms. Phage “life functions”, such as genome replication and synthesis of capsid components, are restricted to occur within infected cells. Outside of the host phages are regarded as metabolically inert, unable to carry out neither biosynthetic nor respiratory functions.
Phages intrigue by their simplistic organization and submicroscopic sizes. These infectious particles consist of a single- or double-stranded nucleic acid genome (DNA or RNA), enveloped in a protein structure (capsid). Current taxonomy and classification of bacteriophages rely on the type of nucleic acid genome and phage morphology, physiology (temperate and virulent life cycles) and genomics. Taxonomy of viruses is supervised by the International Committee for Taxonomy of Viruses (ICTV) that imposes rules for names and writings.
|Cottage cheese, Cheddar,|
|Tvarog, blue cheese|
|Butter milk, fermented cream, butter|
|Mozzarella, Pizza cheese|
|Masdamer, Gouda, Edam, Tilsitter, soft mould ripened cheese, quark, fermented milk beverages|
Lactococcus lactis subsp. lactis var. diacetylactis
Leuconostoc mesenteroides subsp. cremoris
|Mozzarella, Swiss, stabilized soft mould ripened cheese|
|Fermented cream, fermented milk beverages|
Lactobacillus rhamnosus, Lactobacillus acidophilus
Lactococcus lactis subsp. lactis var. diacetylactis
Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis
|* seldom applied in cottage cheese, ** texturizing strains|
The majority of known viruses are bacteriophages, which infect cells of Eubacteria and Archaea. It is also accepted that most phages (96%) isolated so far belong to one taxonomic order of
Bacteriophages, although simple in organization, are the most diverse life forms in the biosphere. Their apparent heterogeneity is reflected by various features – both morphological as genetic, and their persistence on Earth, estimated as high as 1031, outnumbers by far their bacterial hosts . Phages inhabit various niches, like oceans , thermal waters , gastrointestinal tract  and superficial ecosystems created by man, including fermentation tanks in dairy industry . Hence, their impact on the microbial world cannot be underestimated.
Bacteriophage genome structure, indicating linear and double-stranded characteristics of the DNA molecule, containing or not cohesive ends and sometimes presenting terminal redundancy and circular permutation, describes the general feature of LAB phage genomes.
4. Molecular mechanisms of phage infection of LAB
To enter the host, phages firstly come in contact and adsorb to the bacterial cell wall. The adsorption process has been well studied in Gram-negative bacteria, where it was found that two components are involved in the phage-host interaction. One of them is a receptor located in the bacterial cell envelope (membrane or wall), whereas the second component, called the receptor binding protein (RBP), is presented on the phage surface. RBP is responsible for recognition and binding of the phage particle to the bacterial receptor . In the first stage of phage infection, the RBP protein recognizes and binds to a suitable sugar receptor. However, such binding is reversible and thus, the initial phage-bacteria interaction does not ensure commencement of a successful infection event. In contrast to this, in the second stage, a stable phage attachment to the bacterial cell occurs due to an irreversible binding between proteins located on bacterial and phage surfaces . Both stages of adsorption are observed in Gram-positive bacteria: phages that attack
5. Phage sensitivity of LAB starters used in dairy industry
Virulent phages of
6. Defense mechanisms of lactic acid bacteria
It is well documented that lactic acid bacteria evolved defense systems against bacteriophages, which allow them to survive in an environment full of their predators. These anti-phage systems have been organized into five groups depending on the manner by which they operate: (i) inhibition of phage adsorption, (ii) blocking of phage DNA injection, (iii) restriction modification systems, (iv) phage abortive infection systems, and finally, the most recently described, (v) CRISPR/
6.1. Inhibition of phage adsorption
Basic mechanisms of inhibition of phage adsorption to the bacterial cell are associated either with physical masking of the receptor or with changes in its structure, or even with its absence in the cell envelopes . Lack of a functional receptor might be due to spontaneous mutations in the genetic material, leading in turn to bacteriophage insensitive mutants (BIM). A good illustration of the BIM phenomenon is a lactococcal mutant in the chromosomally-encoded
Mechanisms preventing phage adsorption are not only mediated by the bacterial chromosome, but also by acquired plasmids. The best documented plasmid-encoded mechanisms of inhibition of phage adsorption rely on either direct synthesis of cell surface antigens or the production of extracellular carbohydrates. Of the two modes of action, the former reveals phage specificity, whereas the latter seems to restrict access to the bacterial cell for various harmful factors, including bacteriophages . Studies carried by Tuncer and Akcelic demonstrated that a 28.5-kb plasmid, isolated from
6.2. Blocking of phage DNA injection
After phage binding to the receptor, phage DNA is introduced into the bacterial cell. In the cytoplasm, phage genetic information is amplified and consequently progeny particles are produced. However, studies of Watanabe on the interaction between phage PL-1 and a
6.3. Restriction modification systems
Following successful injection of DNA, phage infection might be completed or hindered by the presence of restriction modification systems (RM). RM systems comprise two activities represented by the following enzymes: endonuclease (restriction) and methyltransferase (modification) . Simultaneously, both activities are specific to the same target sequences. The endonucleolytic activity is responsible for degradation of invading foreign DNA, including phage DNA, which lack a unique methylation pattern, while the methyltransferase activity protects the host DNA against degradation by introducing a methyl group into a specific nucleotide of the target site . In detail, phage DNA usually reveal different methylation patterns than those recognized by innate RM systems. Unmethylated target sequences are significantly susceptible to endonucleolytic attack, resulting in DNA degradation . Such mode of action guarantees that the presence of RM systems limits phage proliferation in the cytoplasm, causing no harm to the cell. RM systems are classified into four groups, based on their molecular structure, co-factor requirements, sequence recognition and cleavage position [34-36].
6.3.1. Type I RM
Type I is the most complex RM system in terms of genetic organization and biochemical activity. It is composed of three different
Besides the complex structure of this multifunctional enzyme, also structure of the recognized sequences and cleavage position are the distinguishing features of type I RM systems. Type I RM enzymes specifically recognize asymmetric and bipartite sequences. These non-palindromic DNA sequences consist of two specific components, one of 3-4 bp and the other of 4‐5 bp, separated by a 6-8 bp non-specific sequence [34,36-37]. The innate methylation state of the target sequence determines the activity of the multifunctional R2M2S1 enzyme. When the target sequence is methylated or semi-methylated (e.g. just after replication), the enzyme will exhibit activity of a methyltransferase, which completes DNA modification. In contrast, if the holoenzyme binds to an unmethylated recognition site, DNA translocation past the DNA-enzyme complex occurs in an ATP-dependent manner [35,38]. In spite of DNA translocation, the enzyme remains bound to the target site. DNA is cleaved at a position, where either collision with another translocating complex has appeared or translocation is halted due to the topology of the DNA substrate. Consequently, type I restriction enzymes cleave DNA randomly at a nonspecific site, far from the recognition sequence .
Interaction between subunits, leading to formation of multifunctional enzymes as well as interaction of resultant enzyme molecules with DNA, are determined by the structure of the HsdS subunits. HsdS subunits consist of regions, which amino acid sequences are conserved within an enzyme family, and two independent target recognition domains (TRD) that share low level of amino acid identity [34,39]. TRDs are involved in target sequence recognition, each TRD recognizes one-half of the split target site and is responsible for DNA binding. Since TRDs are highly variable, they recognize multiple target sequences, and thus, provide a variety of phage resistance types [34,36,39]. The central domain, located between two TRDs, is responsible for interaction with one HsdM subunit. Other conserved regions located at N and C termini have been proposed to form a split domain, which makes contact with a second HsdM subunit [35,37].
Type I systems have been further classified into four families based on genetic and biochemical criteria, such as: gene order, identity at amino acid level, complementation assay and enzymatic properties. RM systems belonging to type IA, IB, and ID are only chromosomally-encoded, while most complete type IC systems are either chromosomal or carried on large conjugative plasmids . Additionally, numerous small plasmids carry the
Among LAB, type IC systems seem to be most widespread. Type IC RM loci of both
6.3.2. Type II RM
In contrast to type I, type II RM systems are structurally the simplest of all restriction modification systems. They are generally encoded by two genes, but the key defining feature of this RM type is the independent activity of restriction and modification enzymes . Methyltransferase is active as an asymmetric monomer, requires only AdoMet, and recognizes the same target sequences as the cognate endonuclease. In contrast, restriction endonuclease is a homodimer and requires divalent Mg2+ cations for proper activity. Endonucleases generally recognize a palindromic 4-8 bp DNA sequence and cleave within or in a fixed distance of the recognition site. In contrast to type I, ATP has no effect on the cleavage activity of type II endonucleases .
As this RM type is more heterogeneous in respect to endonucleolytic activity than originally thought based on their structural simplicity, the described mode of action refers mainly to typical (orthodox) type II endonucleases .
Apart from the orthodox type (called IIP ), type II restriction enzymes have been categorized into the following subclasses: IIA, IIB, IIC, IIE, IIF, IIG, IIH, IIM, IIS and IIT. Endonucleases of these subclasses differ in structure of the recognized sequence (asymmetric or symmetric), cleavage positions and cofactor requirements. Type IIA endonucleases behave similarly to the orthodox class, but recognize asymmetric sequences . The unique feature of subclass IIB refers to the cleavage position. These endonucleases cut DNA from both sides, which results in complete extraction of the target sequence from the DNA molecule . Subclasses IIC and IIE have both modification and restriction domains present in one polypeptide. Additionally, class IIE endonucleases interact with two copies of their recognition site, one copy being the target for cleavage, the other serving as an allosteric effector . Similarly to subclass IIE, class IIF restriction enzymes interact with two copies of their recognition sequences, but cleavage occurs at both sequences. Type IIG restriction enzymes seem to combine properties of both IIB and IIC subclasses. The methyltransferase activity of class IIG, like IIB, is stimulated by AdoMet. The main similarity between IIG and IIC is that they both have restriction and modification activities located on one polypeptide chain [45,47]. Subclass IIH, represented by the AhdI system, appears to be a novel RM system due to its genetic organization resembling that of type I. As in type II systems, the AhdI endonuclease is encoded by a single gene; on the other hand, similarly to type I, its cognate methyltransferase forms a complex consisting of two modification and two specificity subunits [44,48]. Subclass IIM is at the opposite extreme from other type II subclasses as it recognizes and cleavages methylated target sequences. The key distinguishing feature of type IIS is the cleavage position outside of the recognition sequence at a defined distance . Subclass IIT is an example of a variation in the typical genetic organization of type II RM systems, as the endonuclease is composed of two different subunits. Moreover, some IIT endonucleases function not only as heterodimers, but also as heterotetramers [44-45].
As enzymes belonging to type II systems are the most abundant and mainly encoded on plasmids, they can be acquired by the bacterial cell through plasmid transfer events. Therefore, a question arises as how to protect the host cell against an incoming endonuclease. In many cases, each gene of the type II RM system has its own promoter. Thus, a delay in appearance of the endonuclease activity is regulated at the transcriptional level. The lactococcal LlaDII RM system is a good example which illustrates this type of regulation . At the initial stage of establishing in the host cell, the LlaDII methyltransferase is overexpressed, whereas the restriction enzyme is produced in small amounts due to the weak constitutive expression of its gene. On the other hand, a permanently high concentration of methylases is an unfavorable circumstance due to possible methylation and therefore protection of the invading phage DNA. The LlaDII methyltransferase contains HTH motifs, which were shown to be engaged in direct interaction with its promoter sequence, causing silencing of its own gene expression .
6.3.3. Type III RM
Unlike types I and II, type III systems are less spread among lactic acid bacteria. The
Lactococci have been found to possess three types of RM systems: type I, II and III. Based on genomic sequence data, it is evident that RM genes are both chromosomally- and plasmid-encoded. However, a variety of RM determinants is generally associated with plasmids . In contrast, very few phage defense mechanisms have been described for
6.3.4. Type IV RM
To date, no type IV RM systems has been distinguished in lactic acid bacteria. It is highly likely that in the future members of this class will be discovered in LAB. For that reason as well as from the evolutionary point of view, the type IV RM system is worth mentioning. A fusion of genes coding for Mod and Res subunits of type III systems was a key step for evolution of type IV RM . The resulting endonuclease (revealing also methyltransferase activity) has an asymmetrical recognition sequence and cleavage occurs at a fixed distance from the recognition site, like for the type IIS enzymes. On the other hand, this endonuclease requires AdoMet, which distinguishes it from type II endonuclease activity. Therefore, taking into account the enzymatic features of model type IV
In summary, it has been well documented that phage restriction-modification systems are widely spread among lactic acid bacteria. Nevertheless, comparative genomics of LAB demonstrated that bacteria representing different niches vary in the presence of restriction-modification genes. The lack of RM systems is a common feature for LAB isolated from the gut, whereas the presence of RM genes is a typical feature for dairy species. Therefore, it was proposed that genes constituting the restriction-modification systems, together with certain genes of sugar metabolism and the proteolytic system, constitute “a barcode” of genes, which can indicate the ability of the microorganism to occupy either dairy or gut niches .
6.4. Phage abortive infection systems
When the RM systems fail in protecting the bacterium against invading phage DNA, initiation of the phage propagation cycle occurs. However, proliferation of progeny particles might be dramatically limited due to systems that abort the infection at various points of the phage cycle. Abortive infection mechanisms (Abi) have different targets in the cell. They are able to interrupt phage DNA replication, transcription, protein synthesis, phage particle assembly or induce premature cell lysis [17,58]. The Abi mechanisms have been found in many bacterial species, including
Abi systems reveal a variety of modes of action. However, in many cases, mechanisms of action of the individual systems were not fully elucidated. Some Abis, like AbiA, AbiD1, AbiF, AbiK, AbiP and AbiT, have been found to interfere with DNA replication, whereas AbiB, AbiG and AbiU arrest mRNA synthesis or have a negative impact on stabilization of transcripts. Haaber and colleagues presented that the AbiV system strongly affects translation of both early and late phage proteins, shortly after infection. Based on this observation, it was concluded that the AbiV system arrests the bacterial translation apparatus . AbiE, AbiI, AbiQ and AbiZ systems affect maturation of phage particles [59,65]. The AbiZ system, identified in 2007 by Durmaz and Klaenhammer, induces premature lysis of phage‐infected cells, resulting in the release of the developing phage particles before completion of the maturation process. The timing of phage lysis is controlled by the phage holin protein; thus, AbiZ might interact cooperatively with the phage holin or with a holin inhibitor to make it active prematurely .
While the mechanism of cell death in the AbiZ system is self-explanatory, in case of other Abi systems is poorly elucidated. The most likely explanation for this phenomenon is that Abi proteins interfere with processes essential not only for phage, but also for bacterial development; therefore, death of individual bacterial cells is always observed following activation of the Abi systems [17,58-59]. As a consequence, release of progeny particles is limited and the bacterial population survives. Hence, the Abi systems constitute a barrier against bacteriophage proliferation, in which “altruistic suicide” of infected bacterial cells provides protection of the whole uninfected population [17,58].
Another naturally-occurring distinct phage defense system recently described in Prokaryotes is CRISPR/
The mechanism of CRISPR/
6.6. Engineered defense systems
Besides the naturally-occurring defense mechanisms against recurrent phage infections (discussed above), new methods involving molecular techniques are designed to combat phages. The constantly growing knowledge on phage development and their genome sequences allows currently to develop engineered defense systems, which are otherwise not encountered in nature (for review see also: ). The idea of such systems relies on engineering bacterial strains in a way which impairs genes vital for phage development, e.g. phage replication proteins or other replication factors. Moreover, identification of homologues of these crucial genes within multiple phage genomes allows creating broad-range phage defense systems. As presented below, numerous studies deliver clear evidence that such engineered systems provide efficient protection against phage infections. The following parts of this chapter will delineate each of these systems in more details. Studies on developing engineered systems for lactic acid bacteria were performed in most part in
6.6.1. Antisense RNA-based phage defense systems
Bacterial-engineered expression of antisense RNA directed against phage transcripts has been described as one of the most efficient phage defense systems. The mode of action of such RNAs is hybridization to phage sense strand RNAs upon infection. By these means the system interferes with the phage life cycle, inhibiting translation of essential phage genes or degradation of their mRNAs .
An example are systems developed in
To test the efficiency of the Sfi21-type module antisense RNA system, constructs expressing antisense RNA cassettes of different length were introduced into
Similar systems were also developed in
Current data allow to conclude that the most effective antiRNA-based phage defense systems, apart from some exceptions, are those which target: (i) genes vital for phage development (e.g. involved in synthesis of phage DNA), (ii) preferably early-expressed phage genes, (iii) genes expressed at low levels, (iv) genes which respective transcripts are unstable [73,79]. Sequencing of novel phage genomes and development of comparative genomics allows identification of other conserved phage genome regions that could serve as potential targets of antiRNAs.
6.6.2. Origin-derived phage-encoded resistance
Defense systems that employ elements derived from lytic phage genomes are termed phage-encoded resistance (PER). One type of engineered PER systems is based on the origin (
One of the first phage origin-derived systems developed was for
More recently, a similar origin-derived phage-encoded resistance system was developed for
Development of analogous systems for other lactic acid bacteria involves identification and functional characterization of
6.6.3. Superinfection immunity and exclusion
During the lysogenic life cycle of temperate phages, the lytic module is inactive due to the activity of the CI repressor. However, certain prophage genes - the superinfection-immunity (CI-like repressor) gene itself and the superinfection-exclusion gene, are actively expressed. Both functions were determined to provide protection to the lysogenic host against phage superinfection. Application of these genes to create engineered phage defense systems is yet another strategy of protecting bacterial cells from incoming infections. Multiple bacterial genomes carry prophage-derived sequences, which can count up to 10% of the total genomic content of the cell. Therefore, despite the fact that phage-related sequences are a burden for bacterial cells, they are also believed to provide some advantage to the host by increasing its fitness.
Genomic studies in
In contrast, superinfection exclusion genes are not engaged in maintaining the lysogenic state, yet are also active during the lysogenic cycle. Experiments based on expression of the
A superinfection exclusion system was also developed in
Putative superinfection exclusion genes seem to be widespread among prophage-containing lactococcal and streptococcal strains and localized in the same genomic region limited by repressor and integrase gene from each side. Although
Superinfection exclusion and immunity genes in natural conditions can also be provided by defective prophages. The nature of defective phages is that they cannot be efficiently induced by environmental factors; hence, cured from the host strain. Such lysogenic lactic acid bacterial strains (particularly
6.6.4. Phage-triggered suicide systems
Phage-triggered suicide systems rely on expression of toxic elements under the strict control of phage-inducible promoters. Such specifically engineered systems most closely resemble the naturally-occurring abortive infection systems, which trap the phage within infected cells and lead to programmed cell death. Upon phage infection, host cells are lysed, disabling at the same time phage propagation and the concomitant spread of the phage. In effect, the uninfected bacterial population is saved (for details see: 6.4. Phage abortive infection systems). Suicide systems are based on three genetic components: (i) a lethal gene cassette, (ii) a phage promoter induced only after phage infection, and (iii) an appropriate vector, providing sufficient amount of the lethal gene product.
Such system, based on an inducible plasmid strategy, was created for
6.6.5. Subunit poisoning
The subunit poisoning system is an engineered phage defense strategy that relies on expression
An example of such system is based on the CI-like repressor of lytic
During the study resistant phages were also detected. Sequence analysis studies within the genetic switch regions revealed alterations in their operator sites, which impaired binding of the Ф31-derived CI repressor.
Another example of subunit poisoning phage defense is a system developed in
Overall, subunit poisoning is an approach that is believed to constitute a broad phage defense system, as it was shown to be effective against more than one lytic P335-type phage. In this aspect, it differs from the earlier described superinfection immunity systems, where expression of phage repressor genes from phages of various lactic acid bacterial species (e.g.
6.6.6. Host-factor elimination
Eliminating a genetic element from the genome of starter bacteria to obtain phage-resistant strains is yet another strategy of engineering a phage defense system. This approach can target different stages of the phage life cycle, which are often host-dependent, e.g. phage injection dependent on host membrane proteins, host factors necessary for phage DNA replication.
Among methods identifying such host-encoded factors is random mutagenesis using the pGhost::IS
Other host factors that were suggested to be efficient targets for developing phage defense systems are auxotrophic genes. Pedersen et al. developed a strategy of impairing phage replication in an industrial
7. Problem of phage contamination in dairy industry
There are no commercial LAB cultures available which would be completely insensitive to all phages. Even when a starter culture that is launched on the market appears to be phage resistant, phages are detected usually after a certain period of use.
Phage contaminations in dairy plants can cause 3 main serious drawbacks:
problems in obtaining expected technological parameters and product quality consistence
staff stress, decrease of motivation and engagement, irregular working hours, staff economical consequences, job resignation
financial losses (failed production, non-standard product, lower unit price, delayed deliveries, customer losses).
8. Phage detection in dairy industry
8.1. Simple tools for phage detection at the dairy plant level
A simple test assessing acidification activity of currently used starter cultures on a daily basis can be used successfully to monitor phage contaminations in dairy plants. Briefly, a cheese whey sample from the last production vat of the shift is collected and, before use, sterilized by filtration (0.45 µm filter-pore size). In the case of dairy beverages, a sample of the final product, before its filtration, is clarified with addition of lactic acid and centrifuged. Processed pasteurized milk or sterilized milk reconstituted from powder is inoculated in duplicate with starter cultures (including a phage alternative culture) at a standard dosage. One sample of each culture is inoculated with a whey filtrate (usually 1-2%) and the second one - with a temperature sterilized whey filtrate. After incubation (the temperature and time depend on the culture and process), the pH of the milk is measured. When the pH of the milk containing the filtrate is 0.2 units higher in comparison to the sample containing the sterilized filtrate, it indicates that phage contamination is rather high and phage-unrelated culture rotation as well as disinfection with higher concentrations of active substances should be recommended.
To avoid direct measurements of pH, bromocresol purple (100 µg ml-1) as a pH indicator may also be used. The test lasts around 6 h, for mesophilic starters, and 4 h, for thermophilic cultures. When pH of the milk drops below 5.4, the indicator turns from purple to yellow. If, at the same time, the color of the sample containing the non-sterilized filtrate becomes green or purple, it means, with high probability, that phages are present and may adversely influence the fermentation process .
Another approach of phage detection is continuous monitoring of pH during fermentation processes conducted in vats or tanks with short time intervals and plotting the data on a graph. Even in the case when delay of the fermentation process is not observed, but the graph shows an irregular shape not related to temperature deviation, phage contamination is suspected (Fig.1). However, in this method a delay in acidification can also result from other inhibitors than phages (e.g. antibiotics, detergents) present in the sample.
8.2. Routine service at culture supplier level
The most common and most useful method of phage enumeration is the plaque assay. The method is quite old and was first described by d’Herelle shortly after the discovery of bacteriophages. Currently it is used in many labs with some modifications, but its principle has not changed . The most common, practical, cheap, without using large numbers of plates and sufficiently accurate method in the dairy industry is the semi-quantitative spot test method. Using this approach, results are available after 24-48 h. The method is well suitable for detection of phages of pure lactic acid bacterial strains at relatively low levels (< 100 phages ml-1). Plague assays allow detecting the presence of phages as well as determining the number of phages in dairy samples against all individual strains present in the applied defined cultures. In case of phage contamination in a dairy plant, the method is a good tool for selecting the best phage-resistant alternative cultures. The method can also be used for hygiene monitoring by enumeration of phages in samples collected from critical places if the plant. For dairy culture producers, permanent phage monitoring can identify strains which are most sensitive in defined cultures. These strains can be systematically replaced with more phage resistant strains. Semi-solid medium supporting bacterial growth is used for multiplication of strains in form of a smooth opaque layer or lawn on the medium surface using standard Petri dishes. Serial dilutions of phage solution previously sterilized with a filter are placed (5-20 µl) on the surface of the opaque layer. When a single phage particle develops on a recipient bacterial lawn, it forms a plaque (clear spot, no bacterial lawn) visible to the naked eye. This plaque results from the destruction of bacterial cells by the phage progeny. Growth of the plaque is limited by slow diffusion of the phage in the semi-solid medium and bacterial cell growth stops, so phage growth is also inhibited due to the fact that host cells support phage growth. No visible plaques on the plate mean that the sample is not contaminated by phages. Large clear zones (no separate plaques) on the plate indicate with high probability that the level of phages is rather high and further dilutions of the sample are required to precisely determine the phage titer. The presence of a plaque means that: i) the tested sample contains phages; ii) the phage is virulent against the tested strain; iii) the strain is sensitive to the phage. Each phage particle that gives rise to a plaque is called a plaque-forming unit (PFU). One plaque corresponds to a single phage particle and phages can easily be counted. In result, the number of PFUs corresponds to the viable phage concentration in a given sample volume.
8.3. Sensitive methods (including ELISA and molecular DNA techniques) at the level of academic or innovation labs
Plaque assays and acidification tests are microbiological methods that are economically accessible and sensitive enough for detection of phages in the dairy industry. These techniques are time consuming, but provide many practical data for both dairy plants and starter producers. The polymerase chain reaction (PCR), ELISA and flow cytometry-based methods have been designed for detecting phages and are often used to complement microbiology tests. However, they have still many drawbacks to be applied for routine analyses in the dairy industry .
PCR-based methods detect virulent and non-virulent phages; thus, microbial methods should be used in parallel to precisely distinguish the virulent phages. PCR-based methods can also be too expensive and too specific (only phages targeted by specifically-designed primers are detected) for routine experiments. However, PCR is a fast method able to confirm the presence of bacteriophages within 30 minutes and can be applied to determine the potential utility and quality of big batches of milk. At the same time, the method could be handy in finding niches of phage accumulation, in order to reduce their impact in dairy fermentations [105-108].
ELISA techniques use for phage detection antibodies which are highly specific against structural proteins of phage capsids. Due to the wide phage diversity in the dairy environment, development of several antibodies detecting various groups of phages was required. ELISA is regarded as a highly useful method for monitoring specific phages in the dairy environment, but a single assay cannot be used to detect phages with different structural proteins. For this reason, the sensitivity of an ELISA method to detect phages in dairy a sample is rather low.
Flow-cytometry can also be used for detection of phages in dairy samples by discriminating the phage-infected cells from non-infected based on cell morphological changes leading to lysis. Running on the flow-cytometry of samples containing phages gives a broad distribution of cell mass (wide peak), which demonstrates the presence of both lysed and live cells, while non-infected samples give narrow peaks. Flow-cytometry allows detection of phages in real time, but expensive equipment and well-trained staff needed to perform the assays limits application of this technique in the dairy industry .
9. Sources of phage contamination
In dairy plants phages can originate from a variety of sources. The prime importance is to identify the potential sources of phage contamination and limit their entry to the fermentation process.
9.1. Raw milk
The most probable source of virulent phages is raw milk. LAB phages occur naturally in raw milk at low titers (between 101‐103 PFU ml-1) and constitute a continuous supply of bacteriophages in dairy plants [109-110]. Phage concentrations in raw milk also depend on conditions of collecting, handling and storing of milk by the supplier (farm), on transport to the plant and, finally, handling of the milk in the plant itself. For example, reverse osmosis used to concentrate raw milk at a farm can impact the level of phages detected in milk. Almost 10% of 900 milk samples examined from various geographical areas in Spain contained
9.2. Milk powder and whey protein concentrates
Reconstituted milk from powder is used in many countries for yoghurt, fresh cheese (tvarog and quark) and even maturated cheese production. Also whey proteins are used to standardize milk before the fermentation process or to improve the taste and texture as well as the nutrient value of the final product. Recently, the modern technology of milk powder and whey protein concentrate production applies often lower temperatures of treatment than during traditional technologies. Both milk powder and whey protein concentrates can be sources of high temperature-resistant phages and can influence the quality of the final product [111-112]. For separating whey proteins, ultrafiltration or/and microfiltration are more frequently employed. Applied separation processes result in higher concentrations of phages in the permeate or the retentate. Depending on which fraction is used in subsequent processes, different concentrations of phages in whey protein samples can be detected.
9.3. Starter cultures
The starter culture itself can be a source of phages, when strains contain temperate phages. Temperate phages are incorporated into the bacterial chromosome and their genome replicates in synchrony with the bacterial genome. Prophages are carried in many LAB strains. The analysis of bacterial genomes revealed that prophages are more widespread than previously considered [113-114]. Phages may be induced from lysogenic to lytic form by the manufacturing conditions. Serial subculturing of temperate phages in milk may result in their replacement by a virulent mutant. Prophage induction from multiple lysogenic starter culture strains has the potential to influence fermentation. Induction can occur under stress conditions, such as heat, salts, acidity, bacteriocins, starvation or UV [115-116], and can also occur naturally with a frequency of even up to 9% . Starter culture producers make huge efforts to eliminate strains containing prophages using a screening assay for strain lysogeny. Usually, easily lysogenized strains are difficult to find in defined strain cultures. The main source of lysogenic strains are undefined cultures, which are still commonly used (for example, kefir grains). This is due to two main reasons: i) the exact strain composition of these starters is unknown; ii) elimination of lysogenic strains from undefined culture is very difficult.
The one of the most probable sources of virulent phages is the dairy plant environment. Phages are commonly present on working surfaces. For propagation, phages need the presence of their bacterial hosts, in this case lactic acid bacteria. Due to this fact, they are usually found in places where conditions for LAB development are favorable. The most common sources of phage contaminations are valves, crevices and “dead ends” (difficult cleaning and disinfection places) of production lines. Also, the formation of biofilms on dairy equipment can lead to serious phage problems. Moreover, phages were detected at high levels on various equipments and objects found in cheese plants, such as walls, pipes, door handles, floors, office tables and even on cleaning materials . Raw milk handling, cheese milk processed in open vats and whey handling can lead to spreading of phages in the air. Phage aerolization can occur during air displacements around contaminated places (fluids or surfaces) or by liquid splashes. Virulent phages can circulate through the air far away from their aerosolization source due to the ability to bind to small particles (< 2.1 μm) . Taking into account high levels of phages detected in the air, it is hard to precisely determine whether phage propagation already took place or if it is likely to occur. Concentrations of up to 108 PFU per m3 of air have been detected in a cheese manufacturing plant in Germany; however, mainly in specific areas of the fermentation line [119-120].
10. Phage problem frequencies and consequences depend on product portfolio
Fermentation problems in the dairy plant can be related with: low starter activity, fermentation conditions (e.g. temperature fluctuations), milk composition (year, season, occurrence of mastitis, mineral levels, lactation period, microbial and enzymatic composition), presence of inhibitors in milk (antibiotics, detergents) and phage infections.
However, phages are the primary source of fermentation problems in the dairy industry. Bacteriophages can cause great economic losses due to fermentation failure in dairy plants. About one third of the annual world production of around 500 million tons is converted into fermented products. Two thirds of all processed milk is fermented by
10.1. Fermented milk beverages
Among dairy products, the least phage affected are fermented milk beverages (yoghurt, kefir, butter-milk, Actimel®-like products, etc.). There are many reasons behind this phenomenon. Milk for beverage production usually undergoes treatment at temperatures much higher than in cheese manufacturing. Moreover, some drinking yoghurts are produced from UHT milk. Beverages are made in relatively aseptic conditions, including more and more aseptic inoculation systems, where the fermented product is minimally exposed to the factory environment. In spite of that, phage contamination is sufficiently frequent and has become the primary source of fermentation problems in milk beverage production. Phage contaminations in this particular case lead to fermentation delays or inhibition, product alterations in taste and flavor as well as texture properties.
10.2. Ripened cheese
In cheese production the risk of phage infection is very high. A large cheese plant can process more than 500 tons of milk per day, very often in many vats, lasting more than one shift. Pasteurized milk (very often low temperature-treated milk or even raw milk) is used in cheese fermentation and many phages as well as microorganisms remain viable after pasteurization. Contamination, also by phages, increases during curd handling and whey separation in open vats. The consequences of phage infection in cheese production can be: delay or halt in milk acidification, cheese contamination with foreign microbiota, including pathogens, preferential growth of post-pasteurization microbiota, problems in whey separation (syneresis), higher water and lactose content in the final cheese product, abnormal or irregular holes (eyes), or no eyes, and alterations of flavor and texture . To conclude, phage contamination may result in lower quality of cheese or cheese quality suitable only for processed cheese production and, in some extreme cases, complete loss of product.
10.3. Fresh cheese (cottage cheese, quark, tvarog)
Cottage cheese and traditional tvarog productions are the most sensitive processes to phages infection. Fermentation delays in production of cottage cheese lead often to complete loss of the final product. However, symptoms of phage contamination are most visible in production of traditional tvarog, where curd quality depends on the activity of lactic acid bacteria alone (rennet is not used). It is estimated that more than 70% of technological disruptions during tvarog manufacture is related to phage contaminations, which usually lead to the following consequences: delay or halt in milk acidification, curd lamination or its drop to the bottom of the tank or vat (which, in effect, causes problem with curd handling), prolonged process of whey separation due to the loss of the curd syneresis, low tvarog yield, contamination with foreign microbiota, including pathogens, intensive growth of post-pasteurization microbiota, off-flavor and texture alterations of the tvarog .
11. Phage control strategy
As previously stated, phages represent a constant threat of serious economic losses in the dairy industry. Dairy microbiologists have attempted for almost 80 years to eliminate or, at least, bring under better control, bacteriophages that interfere with the manufacture of fermented milk products. Phages rapidly disseminate in dairy environment and are difficult to eliminate. The important procedures for phage control are: adapted factory design, design of starters, cleaning and disinfection, and air control .
11.1. Cleaning and disinfection
The classical operations of cleaning and disinfection are an essential part of milk processing. Cleaning-in-place (CIP) procedures are usually applied in milk processing lines. The basic procedure consists of the following sequence operations: i) pre-rinse with cold water to remove gross residues; ii) circulation of alkali detergent to remove the remaining minor residues (from time to time acidic detergent is incorporated to remove precipitated minerals and milkstone deposits in the following sequence: alkali detergent, water rinse, acidic detergent); iii) rinse with cold water to flush out the detergent; iv) circulation of disinfectant to inactivate residual microorganisms and phages (still in many dairies this stage is not performed in each cleaning cycle); v) final rinse with cold water to flush out the detergent and cooling line . The cleaning process can remove 90% or more of microorganisms associated with the surface, but cannot kill all of them. One of the drawbacks of the cleaning process is that residual live bacteria can redeposit and, in longer periods of time, can form a biofilm. The presence of LAB among the residual microorganisms increases phage risk contamination. The main role of disinfection is to kill microorganisms that survive the cleaning procedures.
|Deptil PA 5||Hypred||Hydrogen peroxide,|
|Sodium hypochlorite||0.1 – 3.0||cool||10 – 20|
|0.1 – 0.35||5 – 40||5 – 60|
|0.25 – 1.0||20||15|
|Desinfect CL||Novadan||Sodium hypochlorite||0.20 – 1.0||5 – 40||10 – 15|
|P-3 Oxonia||Ecolab||Hydrogen peroxide||0.5 – 1.0||ca. 10||5 – 30|
|0.1 – 0.2||ca. 10||5 – 30|
|P3 – Oxysan|
|5 – 30|
|0.2 – 0.5||20 – 60||15|
Polyhexamethylene biguanide hydrochloride
|0.5 – 1.5||50 – 70||10|
|Clarin spezial||Clarin||Peracetic acid,|
|0.2 – 0.5||20||5 – 20|
Disinfection is becoming more and more important in the current strategies used by the dairy industry to limit bacteriophage infections. The virucidal efficacy of disinfectants against bacteria, yeasts, moulds, including pathogens, is well-documented in supplier specifications, but very seldom the information on the efficacy against phages is available. It is wrong to consider that disinfectants active against bacteria will also inactivate bacteriophages . The virucidal activity of commercially available disinfectants is unknown or known only against lab reference phages proposed by the established in 1989
|Deptil Mycocide S||Hypred||Propan-2-ol|
|0.3 – 2.5||RT||5|
|Deptil BFC||Hypred||Laurylamine dipropylenediamine||1.0||20 - 90||5 - 15|
|Johnson Diversey||Amphoteric surfactants|
(amines, N-C10-C16- alkyl trimethylenedi, reaction products
with chloroacetic acid)
|0.5 – 1.0||TR|
|15 - 60|
|50 – 100||RT||5 - 15|
|Divosan Extra VT 55||Johnson Diversey||Benzalkonium chloride|
(CAS No 8001-54-5)
|0.4 – 0.8||RT||60 - 240|
|Suredis VT1||Johnson Diversey||Cationic surfactants (N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine|
Disodium tetraborate decahydrate
|0.5 – 2.0||RT .|
|5 - 30|
|Tego Hygiene 2001||Johnson Diversey||Trisodium nitrilotriacetate|
reaction product of alkylamino acetic acid and alkyl diazapentane (CAS: 139734-65-9)
|1.0 – 2.0||RT|
|Virocid||CID Lines||Benzalkonium chloride|
|0.5 – 1.0||RT||60|
|Eko Javel||PUT Ekoserwis||Sodium hypochlorite|
|0.5 – 1.5||RT||15|
|P-3 Topax 91||Ecolab||Benzalkonium chloride|
(CAS No 8001-54-5)
|0.50 - 1||RT||10 – 20|
|P-3 Topax 99||Ecolab||Alkyl ammonium acetate|
|1.0 - static method|
2.0 - foam method
|RT||10 – 20|
|P-3 topactive DES||Ecolab||Hydrogen peroxide|
|P-3 Monodes||Ecolab||Benzyl alcohol|
|Anthium Dioxide 5% active chlorine||GSG||Chlorine dioxide|
Activator – citric acid
|0.01 – 0.05||RT||10|
|* RT – room temperature, ** exposure time|
CEN committee for harmonizing the method of evaluating the efficacy of disinfectants . Factors influencing the efficiency of a given disinfectant are: concentration, temperature and exposure time. Among them, the most important is the concentration of active substances. Most of disinfectants are less effective against phages in the presence of interfering proteins (milk or whey) or hard water. The virucidal activity of most disinfectants is improved by increasing the temperature and is usually the lowest in cold water. Therefore, at low temperatures and/or in the presence of proteins, disinfectant concentration and/or contact time should be increased. It is always advisable to combine biocides and heat rather than use them separately at extreme conditions . However, no disinfectant will be fully effective when sanitized surfaces are not cleaned and proteins or biofilm-living cells are present . Under certain conditions phage particles may exist as aggregates, which may also impair complete inactivation. Peracetic acid and sodium hypochlorite are the most efficient biocides of the CIP system in the dairy industry; however, literature data indicate that some LAB phages may be resistant to sodium hypochlorite [125,127-130]. Nonetheless, the most recently available disinfectants are a combination of several biocides. Table 2 presents the chemical content of CIP disinfectants and conditions of their use in the dairy industry as recommended by the suppliers.
Disinfectants recommended mainly for surfaces, equipment, hands and shoe sanitization are listed in Table 3. Disinfectants are in liquid, foam or aerosol form, depending on their application. The efficacy of such disinfectants for phage inactivation, especially those based on alcohols, are lower in comparison to CIP disinfectants. Among biocides, particularly ineffective in phage inactivation is isopropanol . However, taking into account a lower number of phages in an environment, it can be sufficient for their elimination.
11.2. Design of starter cultures rotation system based on phage contamination control
Starter cultures are a key factor influencing the diversity of phage population in a dairy plant. Application of undefined multispecies and multistrain cultures was the main strategy to overcome production problems related to phages in many factories (Flora Danica - Chr. Hansen, Probat 505 - Danisco) in the past. One complex culture (e.g. Flora Danica) allowed producing many products: maturated cheese, fresh cheese (tvarog and quark), butter, butter-milk and other mesophilic fermented beverages. Complex multispecies and multistrain cultures are relatively phage tolerant and even upon high phage contamination give products with small deviations that are accepted for marketing. In the past, when dairy plants produced a wide range of products, mainly for the local market, complex undefined cultures fulfilled the expectations of the dairy business.
Modern industrial fermentations increasingly rely on well-defined, direct vat inoculated (DVI), high concentrated (> 1010 cfu g-1) and product-optimized starters, containing from two to five phage-unrelated strains [131-132]. Market share of bulk starters (semi-direct inoculation) diminished very fast in the last two decades and does not exceeded 20% for dairy beverages and 60% for cheese of the total global processed milk. The defined cultures have been widely adapted in large-scale production facilities due to the significant degree of control over fermentation processes and complementary fermentation properties, such as rapid acidification, gas formation, texturization, and development of flavor and aroma compounds. Each defined culture is designed in two or three phage-unrelated options, which can consistently enable producers to obtain high quality standard products. Rotation of defined phage-unrelated cultures is an efficient phage control method. Usually the rotation strategy in big dairy plants is elaborated in tight collaboration with culture suppliers based on individual phage monitoring programs. Ideally, sterilized products or whey samples are delivered on a routine basis at agreed intervals to the phage lab of the culture supplier. In longer perspective, successful cooperation of culture suppliers and users in monitoring different culture rotation strategies allows designing sequences of culture rotation and safe intervals between rotations as well as elaborate the cleaning and disinfection strategy adapted to specific dairy environments (Fig.2).
Rotation strategy of defined multiple strain cultures demands selection of strains resistant to a wide range of phages, which could replace infected strains. This aspect can be a drawback when considering continuous and effective use of this method. Moreover, continual rotation of multiple strains during fermentation processes has an effect on phage co-evolution and was shown to increase phage diversity and their abundance in the dairy environment . It also requires constant selection of starter strains with specific fermentative properties. An alternative is the use of a single, highly specialized phage-resistant strain and its variants carrying phage resistance plasmids obtained from naturally resistant strains. This strategy was termed by Sing and Klaenhammer as the phage defense rotation strategy (PDRS) . The success of designed rotations systems of phage-resistant single strain derivatives is assessed by the Heap-Lawrence starter culture activity test (SAT) performed usually in phage-contaminated milk or whey from earlier cycles . Continuous rotation in repeated cycles of single starter lactococcal strain derivatives, where each carries a different type or a combination of various phage defense systems (e.g. R/M or Abi), has been recognized as an effective method of limiting phages during industrial processes [134,136]. Sing and Klaenhammer have shown that the rotation system of three
11.3. Production organization
An important element reducing the spread of phages in the dairy plant is the organization of production. The control of phage risk in dairy plants relies on development and implementation of a variety of procedures. To keep phages under control one should [5,102,123]:
perform daily tests for phage detection
avoid crossing paths for raw milk, pasteurized milk and whey
reduce the diversity of products made on a given day in one production hall
rotate manufacturing processes
directly inoculate milk with high concentrated cultures
rotate starter cultures
use anti-phage media for bulk starter (BS) propagation
perform aseptic inoculation where possible
use air filtration (HEPA) and positive pressure in production facilities
use positive pressure in fermentation tanks where possible
use steam sterilization of production lines where possible, especially when phage contamination is high
dispose stagnant zones of water, whey, milk and foam from production hall or other liquid pools containing live cultures
clean and disinfect lines, floors, walls, bins and drains used immediately after the process completion
redisinfect lines after longer production break (e.g. weekend, bank holiday, breakdown)
disinfect of small equipment used in milk processing after each use (pH-electrode, temperature sensors, etc.)
use footbaths with disinfecting agents at the entry of production facilities
avoid using the same equipment for raw milk and whey transportation and treatment
separate fermentation and packaging areas
limit personnel path movements (staff in contact with raw milk has no admission to the production facilities)
Plant staff should be aware of the importance of phage control risk, well acquainted with procedures and follow them.
12. Selection of phage tolerant strains
12.1. Classical methods (isolation and selection of phage tolerant strains against the most aggressive phages from the dairy environment)
In order to isolate phage-resistant mutants, a secondary culture method can be used , in which sensitive strains undergo selective pressure of their specific phages. Sensitive strains are inoculated in liquid medium and subsequently infected with suspensions of a selected lytic phage at specific titer. Liquid cultures exhibiting complete lysis are incubated for 24-48 hours (secondary growth). After incubation, bacteria are streaked on adequate solid medium. The grown colonies are consecutively cultured in liquid medium with the same selected phage during at least three rounds. Resultant isolates that are able to grow normally in the presence of the specific phage are considered as true phage-resistant mutants .
Another means of natural selection of phage-resistant strains was developed by Viscardi and colleagues . The approach is based on flow-cytometry technique that senses and selects bacterial cells to which phage particles that have been added to the medium did not adsorb. Two detection methods have been designed, which rely on recognition of either specifically labeled anti-phage antibodies or fluorochrome-stained phages. The presented method is an attractive alternative to other means of isolating phage-resistant strains (described earlier). In the study, several different
The great advantage of the method is its high sensitivity (detection of 2 out 107 cells) and high analysis rates (103 cells per second). As the occurrence of spontaneous phage-resistant cells is rather low in nature, the method allows increasing the level of detection of such mutants. Furthermore, the selected
12.2. BIM system - exposure of sensitive strains to lytic phages (spontaneous mutation in chromosomal or plasmidic genes)
Selection of BIMs (bacteriophage insensitive mutants) is a way to obtain phage-resistant strains without genetic manipulations. The idea of obtaining such cells is to infect a starter strain culture and select for mutants which have sustained phage attack.
This approach has its drawbacks, as it is based solely on the occurrence of random potential mutations in genes coding for receptor materials. The lack of a functional initial receptor for 936- and P335-type phages, such as a polysaccharide, is associated with mutations in genes involved in its synthesis or transport. It is well documented that phage insensitivity of
Apart from altering cell growth, other two features, such as narrow phage specificity and spontaneous reversion to sensitive phenotype, limit exploitation of BIM mutants in industrial applications . However, mutations in the
12.3. Plasmid concept
Among the acknowledged and widely applied methods of obtaining starter strains resistant to phage infections is conjugational transfer of plasmids conferring phage resistance determinants [143-144]. In lactococci, there is a range of bacteriophage defense systems occurring naturally on plasmids (natural, plasmid-encoded phage-resistance systems). Among the plasmid-encoded phage resistance are such defense mechanisms as restriction/modification (R/M) or abortive infection (Hsp+ or other Abi+) (for more details see sections: 6.3. and 6.4.). First studies, which linked the presence of phage resistance mechanisms to plasmid molecules, were simple assays based on isolation of plasmids from resistant strains and their reintroduction into susceptible cells to obtain cells immune to attack by a particular phage. The later discovery of phage resistance determinants encoded on conjugational plasmids attracted great interest of the food production industry. Most of the data on conjugative plasmids conferring phage resistance comes from studies in
Among the first conjugal plasmids discovered in
The plasmid-concept of generating phage-resistant strains has also its limitations. First of all, it should be taken into account that many industrially-applied strains are hard to transform. Furthermore, there is a chance that introduction of new plasmids might destabilize industrially attractive strain properties that are also plasmid-encoded (issue of plasmid incompatibility). Introduction of plasmids transferring phage resistance into the bacterial chromosome could be a way of stabilizing this feature; yet, on the other hand, will demand approval of appropriate authorities. Furthermore, some industrially-exploited lactic acid bacteria species, e.g.
casdefense in LAB