**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.

24 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

(*Oenococcus oeni*) [6].

**classification** 

**2. Lactic acid bacteria used in dairy industry** 

Lactic acid bacteria (LAB) comprise different groups of microorganisms, such as *Carnobacterium*, *Enterococcus*, *Tetragenococcus*, *Vagococcus*, *Weissella* as well as species of genera which constitute the "industrial" core of LAB, like *Lactococcus, Lactobacillus*, *Streptococcus*, *Pediococcus* and *Leuconostoc* [6]. LAB reside in different natural habitats, including healthy and decaying plants, milk and dairy products, oral cavity and gastrointestinal tract of humans and animals. In addition, lactic acid bacteria can grow on meat and wine. These features are used in the production of fermented sausages (*Lactobacillus*, *Pediococcus*) and to improve the organoleptic characteristics of wine

The genus *Lactococcus* is the best characterized food-related LAB. As lactococcal strains are able to grow in milk and transform lactose to lactic acid, they are commonly used as starter cultures in industrial fermentations for cheese production. The ability of LAB to transform raw milk into other products suitable for consumption has been used by man for millenniums. Such long history record of interactions of man with lactic acid bacteria and present knowledge led to assigning these bacteria the GRAS status (generally recognized as safe) [7]. Dairy products and the respective LAB species are gathered in Table 1 based on

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 [8]. Therefore, technological problems in production of cottage and hard

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

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),

specifications and recommendations released by the main culture suppliers.

cheeses caused by bacteriophage infections have serious economical consequences.

**3. Lactic acid bacteria phages – history background, morphology,** 

to carry out neither biosynthetic nor respiratory functions.


\* seldom applied in cottage cheese, \*\* texturizing strains

**Table 1.** Various dairy products and LAB species applied in their production.

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 *Caudovirales* [9]. Bacteriophages within this order contain tails and a linear dsDNA genome. They are further classified into three phylogenetically linked families of: *Myoviridae, Siphoviridae, Podoviridae* [9]. *Myoviridae* phages contain a long and contractile tail, while *Siphoviridae* and *Podoviridae* are equipped with a non-contractile tail, long and short, respectively [10]. Isometric heads are dominating (85%) in the morphology of phages from all three families [11]. It is worth to mention that 61% of known phages are classified into the *Siphoviridae* family, of which most of them infect strains of enterobacteria (906 phages), *Lactococcus* (700), *Bacillus* (380) and *Streptococcus* (290) [9]. Apart from the tailed *Caudovirales* phages, there are others demonstrating filamentous, pleomorphic or polyhedral morphology.

Lactic Acid Bacteria Resistance to Bacteriophage and Prevention Techniques

its interaction is crucial both for establishing the reversible and irreversible contact between the phage and the host. In contrast to c2-type phages, phages representing P335 and 936 groups bind to other various bacterial membrane proteins and have been examined in a lesser extent [22]. After establishing a tight connection, they inject their genetic material inside the host cytoplasm, while the capsid remains outside the cell. Then, subsequent steps of phage infection are effectuated which follows either the lytic or lysogenic life cycle. Phages entering the lytic mode immediately redirect the host replication machinery and metabolic functions to replicate its own genetic material and synthesize phage encoded proteins. In effect, abundant amounts of progeny particles are produced. Phages executing only the lytic cycle are designated as virulent and their infection implicates cell death. Yet, certain phages termed as temperate can lead an alternating existence between a dormant state inside the bacterial cell and lytic growth. These phages can exist in the cell in a latent form for generations, replicating in synchrony with the bacterial chromosome. A dormant form of the phage is called a prophage and leads a lysogenic life cycle in a bacterial host strain, which is regarded as a lysogen. Conversion from the lysogenic life cycle to the lytic

often occurs spontaneously or can be induced by various mutagens (UV, mitomycin).

texturizing strains can lead to serious problems in ensuring quality dairy products.

**6. Defense mechanisms of lactic acid bacteria** 

**6.1. Inhibition of phage adsorption** 

Virulent phages of *Lactococcus lactis* spp. are the most frequently encountered phages in milk plants during cheese and dairy beverages production. Additionally, phages attacking *Streptococcus thermophilus* are often observed in cheese and less distinct in yoghurt manufacturing. Phages against *Lactobacillus* spp. and *Leuconostoc* spp. starter cultures represent a minor problem [23]. Currently, in production of dairy beverages functional *Lactococcus* and *Streptococcus thermophilus* texturizing strains with ability to produce exopolysaccharides (EPS) are commonly used. In nature it is very difficult to find strains with similar rheological properties differing in resistance to phages. Thus, phage contamination of

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/*cas* systems. The knowledge about natural phage resistance mechanisms together with a set of genetic tools were applied to develop also (vi) engineered defense systems that confer higher levels of resistance and/or broader phage specificity.

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

**5. Phage sensitivity of LAB starters used in dairy industry** 

to Lower Phage Contamination in Dairy Fermentation 27

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 [12]. Phages inhabit various niches, like oceans [13], thermal waters [14], gastrointestinal tract [15] and superficial ecosystems created by man, including fermentation tanks in dairy industry [16]. 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 [17]. 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 [18]. Both stages of adsorption are observed in Gram-positive bacteria: phages that attack *Lactococcus lactis* cells bind to specific receptors, mainly sugars, located in the cell wall. It is widely known that rhamnose, glucose, galactose, and galactosamine are compounds with which the phage RBP interacts at the initial stage of adsorption [19]. In the case of *Lactococcus* c2-type phages, effective infection requires interaction between phage and the bacterial protein Pip (phage infection protein) [20]. The Pip protein of *L. lactis* is an integral membrane protein [21] and its interaction is crucial both for establishing the reversible and irreversible contact between the phage and the host. In contrast to c2-type phages, phages representing P335 and 936 groups bind to other various bacterial membrane proteins and have been examined in a lesser extent [22]. After establishing a tight connection, they inject their genetic material inside the host cytoplasm, while the capsid remains outside the cell. Then, subsequent steps of phage infection are effectuated which follows either the lytic or lysogenic life cycle. Phages entering the lytic mode immediately redirect the host replication machinery and metabolic functions to replicate its own genetic material and synthesize phage encoded proteins. In effect, abundant amounts of progeny particles are produced. Phages executing only the lytic cycle are designated as virulent and their infection implicates cell death. Yet, certain phages termed as temperate can lead an alternating existence between a dormant state inside the bacterial cell and lytic growth. These phages can exist in the cell in a latent form for generations, replicating in synchrony with the bacterial chromosome. A dormant form of the phage is called a prophage and leads a lysogenic life cycle in a bacterial host strain, which is regarded as a lysogen. Conversion from the lysogenic life cycle to the lytic often occurs spontaneously or can be induced by various mutagens (UV, mitomycin).
