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The infection of a growing bacterial culture with phage is initiated by the adsorption of the phage to the host cell. The specificity of adsorption of lactococcal phages and the location of phage receptor substances have been studied and has been reviewed (Lawrence et. al., 1976).

 Lactococci have been shown to have different receptor sites that may reside in both the cell wall and plasma membrane. Some phages may be relatively strain specific attacking only one or two strains but others show less specificity and may even attack strains of different subspecies, i.e. a phage may attack strains of Lc. lactis, Lc. lactis subsp. cremoris and Lc. lactis biovariant diacetylactis. Although many strains are resistant to a specific phage because the phage cannot adsorb to receptor sites some strains which allow adsorption are also phage resistant . The resistance of the latter has been attributed to lysogenic immunity or to the operation of a modification/restriction (M/R) system .

It is generally recognised that the ionic environment is an important factor in adsorption. In particular, inorganic salts must be present (Cherry and Watson, 1949) and probably act by neutralising the net negative charges on the host cell and its phage so that initial contact is facilitated. Calcium which is required for the multiplication of most lactococcal phages (Shew, 1949; Potter and Nelson, 1952) is not a specific requirement for adsorption since monovalent cations are apparently just as effective (Reiter and Møller-Madsen, 1963); note some lactic phages do not require calcium for replication. The calcium-dependant requirement for replication has been exploited in the development of phage inhibitory media for phage-free bulk starter production.

The adsorption of some phages to lactococci has been reported to be reduced by rennet, increased temperature or by growth on a different host (Keogh, 1973). Although the differences were often small, the combination of rennet with elevated temperature (37°C) substantially reduced adsorption for several phages.

DNA introduction

The methods whereby phages eject or introduce their nucleic acid into lactic acid bacteria are not fully understood. Because phages without DNA can easily be observed attached to cell walls and that it is obvious that this DNA must have passed from the head through the channel in the phage tail, many authors call this process DNA injection. This terminology is not precise since the majority of lactic phages do not possess a contractile tail and while the use of the term injection is understandable, it is inappropriate for most phages. I prefer to use the term DNA introduction. Calcium and some other divalent cations are generally required for DNA introduction. This has been exploited in phage control as discussed previously.

The cell wall of Gram-positive bacteria, including lactic acid bacteria is fairly thick, ca. 20 nm for lactococci, and this is the first significant barrier that the phage must penetrate if it is to successfully infect its host. Much more is known about how phages for Gram-negative bacteria get their DNA into cells. Some, e.g. T4, T7, PRD1 have been shown to have tail-based lytic enzymes.
 These enzymes are thought to literally 'punch holes' in the cell wall and generate channels for DNA introduction.

Until recently tail based lysins had not been demonstrated in phages for lactic acid bacteria. Kenny et al. (2004) found that a phage for Lc. lactis UC509, designated Tuc2009, had a phage tail lysin and demonstrated that this was required for efficient viral infection. This discovery raises the issue of whether all lactic phages contain tail-based lysins or whether there are a range of mechanisms that phages use to obtain access to the cell membrane.

Early studies of the cell wall of a Lc. lactis strain by Hurst and Stubbs(1969) appeared to reveal holes in the cell wall through which protrusions of the cell membrane were observed. If this is true even for some strains then lactococcal phages may already have direct access to the cell membrane. Note this finding has not been confirmed and it has been suggested that the 'holes' may be artifacts.

Maturation

The intracellular development of phages for lactic acid bacteria is still not fully understood. Early studies reported that the host nuclear material of lactococci was completely disrupted following infection.

From studies with coli phages it is clear that the phage nucleic acid takes over the cell's biosynthetic machinery and phage specified messenger(m)-RNA's and proteins are made. Early m-RNA's code for early proteins which are needed for phage DNA synthesis and for shutting or turning off host DNA, RNA and protein biosynthesis. In some cases the early proteins degrade the host chromosome. After phage DNA is made late m-RNA's and late proteins are made. The late proteins are the structural proteins that comprise the phage as well as the proteins needed for lysis of the bacterial cell.

For many years the limited information available concerning maturation has been obtained from one step growth experiments as developed by Ellis and Dalbruck (1939). These experiments yield information on the duration of maturation i.e. the latent period, and the burst size. The latent period may be defined as the minimum length of time from the adsorption of phage to its host, until the release of newly formed phage particles. The average yield of phage particles per cell is the burst size.

The latent period and burst size of some lactic streptococcal phages have been reported in skim milk at 30° and 37°C (Keogh, 1973; Zehren and Whitehead, 1954). Keogh reported latent periods ranging from 32 to 56 minutes at 30°C and from 32 to 44 minutes at 37°C; burst sizes varied from 2-105 particles at 30°C and 0 - 77 particles at 37°C. Note that some lactococcal phages are temperature-sensitive.

Temperature has a major effect on phage replication. Some phage-infected bacteria do not produce phage at 28°C but do so at 18°C.  Others do not produce phage at temperatures >34°C while certain phages are produced at 37°C but not at lower temperatures.  The existence of phages that replicate at all temperatures permitting host growth must also be recognised.  While a standard temperature of 30°C is often used in studies of phage enumeration the temperatures used in the fermentation process must be reflected in methods used to detect phage if reliable results are to be obtained. See also the section on factors affecting plaque formation.

Lysogenic or temperate phages

Unlike the action of  lytic phage discussed previously, temperate phages do not normally kill their hosts. Note the term temperate should be used instead of lysogenic phage.  Instead the phage enters into a lysogenic relationship with its host in which the phage genome or a replica of it, becomes inserted into the bacterial chromosome. This relationship is very stable and is transmitted to following generations of the host. Phage in this form is known as prophage.

Although the origin of phage active against lactococci is unknown, lysogenic starter culture strains have been suspected for some time as a possible reservoir. There is now an increasing volume of circumstantial evidence that the main source of phage in cheese factories is from the starter cultures themselves. However, lysogenic strains of lactococci present in the raw milk must also be considered as a possible source.

Heat stability of lactococcal phages

Most phages for lactic acid bacteria survive HTST pasteurisation. Generally a heat treatment of 85°C for 15 minutes will destroy most lactococcal phages. However, some phages will survive for 5 min in whey(pH 6.0) or for 2 h (when dried) at 90-95°C in an oven. In view of the high heat treatments required to destroy some phage, heat treatments in excess of 95°C for 10 min should be employed to ensure freedom from phage. Since dried phage preparations are very resistant to heat, thorough cleaning is essential to remove any dried phage particles from starter vessels or equipment prior to sterilisation by heat.

Sensitivity to pH

Hunter and Whitehead (1940) reported that phage could be destroyed by the addition of 2.5% v/v lactic acid to a preparation of phage in milk, at ambient temperature in 5 min. A concentration of 1% lactic acid was less effective and about 24h exposure was required for inactivation. More detailed experiments by the above workers showed that phage destruction was rapid at room temperature at and beyond the limiting pH values of 2.5 and 11.8. Between these values the destructive effect fell off rapidly, and in particular between pH 4.0 and 7.0 there was no appreciable effect over a period of five days.

Nevertheless, it is now known that many phage preparations are rapidly inactivated at pH values below 5.0, particularly below 4.5, when held at room temperature.

Effect of disinfectant and sterilising agents

The effect of some sterilants on phage destruction are shown in Table 1.

Table 1 Efficiency of various sterilants for phage destruction

 

Sterilant

Final concentration in phage-sterilant mixture

%

Time required for complete destruction (room temperature)

Hypochlorite

0.05

Less than 1 min

KMn04

0.25

0.05

0.025

Less than 1 min

Between 1 and 5 min

Not in 2 days

H2O2

3.0

2.5

0.5

Between 15 and 60 min

Between 1 and 24 hr

Between 1 and 24 hr

Formaldehyde

5.0

2.5

1.0

0.5

Between 5 and 30 min

Between 30 and 60 min

Between 1 and 24 hr

Between 1 and 24 hr

HgCl2

2.5

1.0

0.5

Between 1 and 24 hr

Between 2 and 3 days

Not in 14 days

Alcohol

90.0

85.0

80.0

75.0

70.0

Between 3 and 4 days

Between 2 and 3 days

Between 2 and 3 days

Between 3 and 5 days

Not in 6 days

Phenol

2.5

Not in 14 days

Source:  Hunter and Whitehead (1940)

These results which have been confirmed by many workers demonstrate the effectiveness of hypochlorite as an antiviral agent. Note that quite high concentrations of formaldehyde are required to inactivate phage and that alcohol has limited effectiveness.

Phage aerosols are the major vehicle of phage infection in cheese factories. It is obviously important to have information concerning the effectiveness of various sterilants in aerosol form on their ability to destroy aerosols containing lactococcal phages. Hypochlorite is particularly effective, however, it is very corrosive. Organic chlorine-containing compounds e.g. dichloroisocyanuric acid (DCCA) are almost as effective as hypochlorite but are much less corrosive.

Quaternary ammonium compounds (QACs) have been shown to have little anti-phage effect. Iodophors are much more effective than QACs but are of limited practical use because they may stain surfaces.

 
How to cite this article

Mullan, W.M.A. (2002). [On-line]. Available from: https://www.dairyscience.info/index.php/bacteriophages-for-lactic-acid-bacteria-with-particular-emphasis-on-lactococci.html . Accessed: 3 December, 2016.

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