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Bacteriophages for lactic acid bacteria

This section provides links to articles on the discovery, biology, lysins, industrial significance, control, isolation, propagation, storage and enumeration (assay) of lactococcal bacteriophages on this website. Information on other phages is also covered.

It is important to note that while bacteriophage, usually abbreviated to phage, infection is a major cause of poor growth and acid production by starter cultures, that these bacteria may also be inhibited by added substances including antibiotics, sterilant and detergent residues, or free fatty acids produced by or as a result of the growth of microorganisms, and natural often called indigenous antimicrobial proteins. Additionally use of incorrect temperatures during fermentation processes can also cause problems.

Discovery of bacteriophages for lactococci

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.

Whitehead and his colleagues isolated lactic streptococci, known known as lactococci, from the undefined, mixed strain cultures and identified a number of single strains that could be used on their own to produce quality Cheddar cheese.

While the use of single strains of lactococci was initially very successful, instances in which the starter completely stopped producing acid with the resultant complete failure of the cheesemaking process were soon encountered. The New Zealand cheesemaker termed this the 'pack up' phenomenon for obvious reasons. The term 'dead vats' was used to describe the non-acidifying curds in the vats.

Viruses obligately parasitic on bacteria were discovered independently by Frederick Twort in 1915 and Felix d'Herelle in 1917. However, it was d'Herelle who first used the term bacteriophages ("eaters of bacteria") to describe viruses for bacteria.

Whitehead and Cox (1935)* discovered lactococcal bacteriophages as the causal agent of the complete cessation of acid production experienced using single strain starters.

With hindsight it is obvious that the use of single strain cultures had created ideal conditions for phage proliferation, and lysis of starter cells, resulting in catastrophic fermentation failure. Additionally, heat treatment of the cheese -milk had eliminated the natural lactic flora that previously would have produced acid and could have been used as 'a backup' source of acidification. However, the use of single strain starters also facilitated the discovery of phage and the subsequent development of control strategies.

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Properties of bacteriophages for lactic acid bacteria

Phage adsorption

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).

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Characteristics of 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.

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Bacteriophage control in cheese manufacture

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. The 1945 review focused on whey as the vehicle for phage transmission, and on work designed to break the cycle of phage infection. Even in 1945 they recognised the challenge of keeping phage concentrations low in the environment, the need for special facilities to produce phage-free bulk starter in a potentially phage-infected environment, the possibility of raw milk being contaminated with phage because the cans, tankers today, carrying whey, and also used to carry raw milk, could contaminate raw milk. They were also aware that whey separators and 'splashes' of whey produced aerosols and that these would enable phage to become airborne. Off course they were not aware of lysogency and the possibility of phage arising from the starter or lactococci in raw milk.

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Bacteriophage lysins

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.

The presence of phage-induced, cell-wall degrading enzymes in lysates of phage-infected bacteria has now been described for an extensive range of bacteria including Escherichia coli, Staphylococcus aureus, Azotobacter agilis, Aerobacter cloacae, Bacillus megaterium, Micrococcus lysodeikticus, Bacillus stearothermophilus, Klebsiella pneumoniae, Pseudomonas putida, Ps. aeruginosa and for lactic acid bacteria including lactococci, leuconstocs and lactobacilli.

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Effect of lysin-producing phages in dairy fermentations

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.

The effect of infecting paired-strain cultures with ØC2 (W) is shown in table 2. Acid production was markedly inhibited for six of the eight combinations. Extensive replication of ØC2 (W) occurred in all phage-infected cultures. Phage infection did not inhibit acid production when the

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Assay of phage lysins

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.

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Isolation and purification of phage lysins

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 10 pfu/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.

Effect of phage lysin on Lc lactis C10 

To obtain significant purification several purification strategies are generally employed. Ion-exchange chromatography is usually employed at an early stage.

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Enumeration of lactococcal bacteriophages

WHY ENUMERATE BACTERIOPHAGES?

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

 • To determine the level of phage contamination of dairy processing plant.
 • To determine the effectiveness of cleaning and sterilising programmes.
 • To determine levels of airborne phage.
 • To determine if cultures are contaminated.
 • To obtain information on phage/culture relationships.
 • Determine the efficiency of virucidical filters.

Materials tested for phage are generally called phage suspect materials (PSM).  These include starters; raw milk; cheese milk; whey; the clean-in-place solutions and rinse water; pipelines, vat surfaces, valves, cheese-making implements; plant atmosphere; curd and cheese. 

Although the bulk of cheese factories in the UK no longer propagate their own starters, only 17 % in 2000 ( Boyle and Mullan, unpublished),  examination of starters for phage contamination is important for those producing their own bulk starter.  Examination of the milk in cheese vats immediately prior to starter addition is important  since the concentration of any phage present provides a good indicator of the effectiveness of rotations, the insensitivity of cultures to phage and the effectiveness of the CIP system and the level of plant hygiene.

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Plaque formation by bacteriophages

The double agar method as described by Adams(1959) is widely used to enumerate lactococcal and other 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 107-108 CFU/ml, are mixed in about 2.5 ml of molten, 'soft' agar at 46°C.  The resulting suspension is then poured on to an appropriate 'nutrient' basal agar medium e.g. M17 (Terazaghi and Sandine, 1975) to form a thin 'top layer' which hardens and immobilises the bacteria. Refer to figure 1 below.

TPlaque assay using the double agar assayhe basal agar should be free of surface moisture to avoid problems, occasionally encountered, of the soft agar not adhering to the basal agar.  Solid, thermostatically-controlled heating blocks are useful for maintaining the soft agar, see figure 2, at the correct temperature and are more useful than water baths. The assay can also be undertaken by adding phage, host and calcium ions to a sterile test tube at ambient temperature. After several minutes molten soft agar at 46°C is added and the mixture poured on to the basal agar as discussed previously. During incubation usually at 30°C, uninfected bacteria multiply to form a confluent film of growth over the surface of the plate. 

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Factors affecting plaque formation?


How do you get bacteriophages to form plaques?

Abstract
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".

The conditions required to get a newly isolated phage to form plaques have been reviewed. The importance of testing both logarithmic and stationary phage cells, a range of incubation temperatures, replacing agar with agarose, using low strength agarose top agar overlays in initial experiments, media supplemented with Ca++ and Mg++ that do not contain cation chelators, modifications to the double agar assay method including a) changes to the initial assay step so that phage adsorption takes place at ambient temperature  and b) the use of antibiotics and other activators of the host cell’s SOS system are discussed.


Difficulties in getting phages to form plaques 

Phage enumeration using the double agar assay is an essential control test in many quality assurance systems involving bacterial fermentation processes. However, increasingly phages are being used in the treatment of human and animal diseases, to prevent spoilage of fruits and vegetables, to eliminate biofilms in food processing plants, in studies of nano-machines and nanotechnology and in extensive genetic and molecular biology research.

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