Bacteriophages for lactococci

Details: Written by: Michael Mullan

Details: Written by: Michael Mullan

Prior to the early 1930's most cheese was made from undefined starter cultures; species and strain composition were generally unknown and if known initially would change with each subculture.

Dr Hugh Whitehead and his colleagues at the New Zealand Dairy Research Institute realised that if the dairy industry in that country was to produce close-textured cheese, free from taste and body defects and manufactured within a consistent time period that it would be necessary to use standardised starter cultures. They also realised that they needed to prevent problems arising from the growth of 'wild' lactic acid bacteria and spoilage organisms in the raw milk and introduced pasteurisation of milk for cheese manufacture.

Details: Written by: Michael Mullan

Any agent which inhibits starter activity or kills a strain with an essential function e.g. aroma production can have serious detrimental effects on the quality of the product being produced. Infection with bacteriophage is the major single cause of fermentation failure or of problems in fermentation processes utilising lactic acid bacteria.

The major functions of starters in dairy fermentations are shown in table 1. See the section on starters also.

Details: Written by: Michael Mullan

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).

Lactococcal bacteriophages

Bradley (1967), in a classic review paper, summarised the principles of phage morphology and outlined six basic morphological types (fig. 1). The tailed phages, Bradley's groups A-C account for some 96% of all phages isolated to date and as discussed below belong to the order Caudovirales. Only phages in Group A have contractile tails. All tailed bacteriophages have a nucleic acid core surrounded by a protein coat. Phages active against lactic acid bacteria are approximately tadpole or sperm shaped and have a distinct head terminating in a tail with a hollow core.

Phages attacking lactic acid bacteria belong to Groups A, B and C and contain double stranded DNA. Phages in Groups D and F contain single stranded DNA, however, Group E phages contain single-stranded RNA.

Details: Written by: Michael Mullan

The basic principles of phage control in commercial plants have been known since the early 1940s and the pioneering work of Dr Hugh Whitehead and his colleagues in New Zealand. The review by Whitehead and Hunter (1945)* on the measures that were being used in New Zealand to control slow acid production due to phage infection is still of relevance to factory managers today.

Details: Written by: Michael Mullan

Phage release, the final stage in the phage-life cycle, has been extensively studied and is caused, at least in part, by the action of phage-induced hydrolytic or lytic enzymes.

Details: Written by: Michael Mullan

Because phage lysin has a much broader lytic range than phage, infection of paired and multi-strain cultures with a lysin-producing phage has the potential to cause fermentation failure, dead-vats, and consequent economic loss.

Details: Written by: Michael Mullan

While phage lysin has long been suspected of having an important part in phage lysis it has taken techniques using molecular biology to clarify its in vivo role.

Details: Written by: Michael Mullan

The activity of phage lysins can be determined using several methods; turbidimetry or the determination of the change in concentration of some solubilised cell wall component are frequently used.

Turbimetry, where a standardised suspension of cells in buffer is mixed with a sample of lysin-containing material is widely used. The lysin causes lysis of the cell suspension and a reduction in optical density (OD). Enzyme activity can be calculated from the decrease in OD with time.

The following method has been found to give satisfactory results (Mullan and Crawford, 1985a).

Cells were suspended in 0.1 M-K phosphate buffer, pH 6.8, to give an OD at 450nm of 0.62-0.75. Depending on the lytic activity, 0.1-1.0 ml of lysin containing solution was added to 5ml of standardized cell suspension at 37°C. After mixing, OD readings were taken at 30 s intervals over a 2-6 min period. Absorption readings were plotted against time and only values on the linear portion of the graphs were used for calculation.

Details: Written by: Michael Mullan

The first stage in the isolation of phage lysin is the production of lysates containing high concentrations of phage. Because lysin concentration is correlated with phage concentration, this objective can be achieved by obtaining lysates containing >1 x 10 ¹⁰pfu/ml (Mullan and Crawford, 1985a). Information on the production of high tire phage lysates has been discussed previously. The effects of phage lysin on cells of Lc. lactis c10 is shown below. The lysin rapidly removes the cell walls resulting in cell death.

Details: Written by: Michael Mullan

There are many reasons why information on the concentration of bacteriophage in a sample may be required. These include the determination of:

Details: Written by: Michael Mullan

The double agar method as described by Adams (1959) is widely used to enumerate phages. In this method a small volume of a dilution of phage suspension and a small quantity of host cells grown to high cell density, sufficient to give 10⁷-10⁸CFU/ml, are mixed in about 2.5 ml of molten, 'soft' or 'top' agar at 46°-50°C. It is important to avoid over mixing the soft agar since that could result in air bubbles forming in the soft agar and potential misidentification of the bubbles as plaques. The resulting suspension is then poured on to an appropriate 'nutrient' basal agar medium e.g. M17 (Terazaghi and Sandine, 1975) for lactococci to form a thin 'top layer' which hardens and immobilises the bacteria. Refer to figure 1 below.

Details: Written by: Michael Mullan

Abstract

Over 99% of phages detected using microscopy have not been cultured. This article explores factors that influence plaque formation and if addressed may help in phage isolation.

Current data indicate that some 10³¹ bacteriophages exist globally, including about 10⁸ genotypes. Some phages form very tiny or micro plaques. These can sometimes be so small that it is almost impossible to see them. Frequently 'new' phages can be observed using e.g. electron microscopy under conditions where there is strong evidence of a potential host yet it can be very time consuming or in some instances not possible to get the phage to form plaques. Less than 1% of the phages observed using microscopy have ever been grown in culture, this is sometimes called "the great plaque count anomaly".

Details: Written by: Michael Mullan

Phage activity can also be assessed indirectly by measuring culture activity, the premise being that the presence of disturbing phage will inhibit starter growth.

Several methods are available, and include-

Details: Written by: Michael Mullan

How do you isolate a bacteriophage (phage) and obtain a pure phage preparation? This is achieved by plating a phage suspension using the double agar method, and a susceptible host strain, to obtain plaques and further purifying the phage contained within the plaque.

Details: Written by: Michael Mullan

It is frequently necessary to produce and use high titre phage preparations.

This section provides summary information on the production and storage of high-tire lactococcal phage preparations.