This document discusses bacteriophages (phages) that infect lactic acid bacteria (LAB) used in dairy starter cultures. It describes four main mechanisms of phage resistance in LAB: 1) adsorption inhibition through changes to cell surface proteins and polysaccharides, 2) DNA injection blocking, 3) restriction modification systems, and 4) abortive infection systems. It also outlines various methods used in dairy plants to control phages, including phage-inhibitory media, culture rotation, development of phage-resistant mutants, encapsulation of cultures, and improving sanitation.

1. Bacteriophages of LAB: Control
measures and
their significance
Prasanta Kumar Choudhury
National Dairy Research Institute
Karnal, Haryana, 132001

2. Bacteriophages and Dairy industry
• The occurrence of bacteriophage (phage) in dairy
starter cultures was first reported in the 1930s by
Whitehead and Cox (1935) in Lactococcus starter
used for cheddar cheese.
• The existence of phage is a recognized problem in
the dairy industry.
• But the LAB have some phase resistant mechanisms

3. Phage resistance mechanisms of LAB
1. Adsorption Inhibition
2. DNA injection blocking
3. Restriction modification
4. Abortive inhibition
Phage resistant mechanisms of LAB:

4. 1. Adsorption inhibition
Attachment of a phage to the cell surface is a very
specific process, dependent on :
• Phage specificity
• Accessibility of bacterial receptor
• Physiochemical properties of the cell envelope
• The electrical potential across the cytoplasmic
membrane

5. • The surface of Lc. lactis was covered predominantly
by polysaccharides and proteins in the ratio 2 : 1
• The composition/ structure of the polysaccharides
most likely varies between different strains.
• The phages first recognize a polysaccharide before
they attach to a protein located in the plasma
membrane
• The polysaccharides are often composed of
rhamnose, glucose, galactose, and glucosamine and
are covalently linked to the peptidoglycans

6. • An S-layer composed of a protein covered the
surface of L. helveticus strains.
• Change in the amino acid sequence can affect phage
adsorption
• Failure of a phage to adsorb to a host cell may be
due to either the lack of appropriate
polysaccharides on the cell surface or to a physical
masking of the receptor polysaccharide or protein

7. • Polysaccharides may either be excreted into the
environment as exopolysaccharides (EPS) or form
capsular polysaccharides (CPS)
• It has been discovered that production of EPS in
some cases blocks adsorption, but not always CPS
provide bacteria with a higher degree of protection
than EPS

8. 2. DNA injection blocking
• DNA injection inhibition occurs when phage
adsorbs to the cell surface but phage DNA stays
inside the head section and fails to enter the host
cell cytoplasm.
• DNA injection inhibition resulted from an alteration
in plasma membrane components of the host cell.
• This resistance mechanism appears to be rare
(Dinsmore and Klaenhammer, 1995).

9. 3. Restriction/Modification
• An R/M system first recognizes a specific DNA
sequence; then it either modifies DNA by a
methyltransferase or cleaves DNA with a restriction
endonuclease
• Methylation is performed either on adenine or
cytosine located within the recognition sequence by
transfer of a methyl group from S-adenosyl-L-
methionine
• The bacterium methylates its own DNA, thereby
protecting it against restriction

10. • Cleavage by the restriction endonuclease will take place
either within or nearby the recognition site or randomly
Therefore the R/M system can protect bacteria against
foreign DNA such as phages by cleaving invading phage
DNA
• It only requires that the recognition sequence specific
for the R/M system is present in the phage DNA and
that the sequence has not been modified by
methylation
• The DNA of progeny phages that has escaped restriction
by an R/M system will be methylated, and therefore
these phages can circumvent that specific R/M system
and reinfect the bacteria with a high efficiency

11. 4. Abortive inhibition
• Abortive infection is a type of phage resistance
resulting in decreased production of virulent
phages by infected cells but not involving restriction
or modification.
• Abortive infection results in cell death, but because
phage replication is much reduced, the phage
population does not increase sufficiently to affect
culture activity.
• Abortive infection does not induce genetic changes
in the infecting phage.

12. • Most of them are plasmid-encoded. All Abi systems are
characterized by an unusually low G + C content of their
genes (26–29%)
• The AbiA, AbiK, AbiF, and AbiR systems act on the phage DNA
replication
• AbiG, AbiU, and an undefined Abi system encoded by
plasmid pBU1-8 from Lc. lactis subsp. lactis Bu2 inhibits or
delays transcription of phage DNA
• AbiB was shown to prevent phage growth by promoting
degradation of transcripts derived from phage bIL170 (936
species) infection of Lc. lactis subsp. lactis IL1403 starting 10–
15 minutes after infection

13. • In cells harboring AbiQ, the immature concatemeric
form of phage DNA accumulated in the cells. This
suggests that it may be defective and unable to be
processed into mature phages or that genes
involved in phage morphogenesis are affected by
AbiQ
• It has been suggested that one of the late mRNAs or
proteins activates the AbiT mechanism and causes
premature cell death

14. Abortive Infection Mechanisms Characterized from
Lactococcus

15. Problems caused by bacteriophages
• Slowing of starter or starter failure
• Poor acid development
• Decrease in flavor production
• Poor rennet action
• Poor texture formation
Great loss to dairy industry

16. 1. Use of Phage-Inhibitory Media
2. Culture Rotation
3. Development of phage resistant mutants
4. Encapsulation of starter cultures
5. Dairy plant and cleanliness
PHAGE CONTROL
Artificial Phage-Resistance Methods

17. 1. Phage Inhibitory Media
• Growth of phages during production of bulk starter
can be controlled by using phage-inhibitory media.
• These media rely on the ability of phosphate and
citrate salts to bind ionic calcium, thus inhibiting
phagic absorption (Reiter, 1956).
• Phage-control media often contain deionized whey,
protein hydrolysates, ammonium and sodium
phosphate, citrate salts, and other growth stimulants
such as yeast extract (Whitehead, 1993) which
prevents phage proliferation.

18. 2. Culture Rotation
• Culture rotations control bacteriophagic infection by
limiting the length of time that a specific strain or
mixture of strains is used.
• Cultures following each other in the series are
susceptible to different phage types and are therefore
unaffected by phages that may have infected the
previous culture.
• Cultures can be rotated on a daily basis or after each vat
of milk is inoculated.

19. • However, the use of a limited number of cultures at any
one time is recommended to reduce exposure to
prophages and maintain product uniformity.
• Culture rotation does not eliminate phage growth in
cheese milk in vats, but if phage numbers are kept to less
than 10,000 pfu/mL of cheese whey, acid production is
not affected (Huggins, 1984).
• Success of a culture rotation is limited by availability of
phage-unrelated strains with acceptable fermentation
properties. In addition, using many different cultures can
result in lack of product uniformity.

20. 3. Development of Phage resistant mutants
I. Antisense RNA Strategies
II. Utilization of Origin of Replication (PER)
III. Utilization of the Phage Repressor

21. I. Antisense RNA Strategies
• A gene is cloned behind a promoter in its antisense
orientation
• It is anticipated that its antisense RNA transcript
will bind to target sense mRNA, preventing
translation either by destabilizing and making it
more susceptible for degradation by RNases or by
inhibiting loading of ribosomes

22. II. Utilization of Origin of Replication (PER)
• Another approach is based on utilization of the phage
origin of replication
• The method was designated “PER” for phage-encoded
resistance
• When the cloned origin of replication (ori) of the
lactococcal phage Ф50 was introduced into Lc. lactis
subsp. lactis NCK203, the host’s insensitivity to Ф50
infection was enhanced as indicated by a reduction in
EOP and in plaque size
• It was assumed that the cloned ‘per’ locus competes
with normal phage replication.

23. III. Utilization of the Phage Repressor
• Temperate phage have a gene encoding a repressor,
CI, suppressing the expression of the lytic cycle
• Integration of the cI gene of phage A2 into the Lb.
casei host chromosome resulted in stable resistance
against super infection with the A2 phage

24. CRISPR DNA
• CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats)
are loci containing multiple short direct repeats that are found in the
genomes of approximately 40% of bacteria and 90% of archaea.
• Prokaryotic immune system confering resistance to exogenous genetic
elements such as plasmids and phage.
• Short segments of foreign DNA, called spacers, are incorporated into the
genome between CRISPR repeats, and serve as a 'memory' of past
exposures.
• CRISPR spacers are then used to recognize and silence exogenous
genetic elements in a manner analogous to RNAi in eukaryotic
organisms

25. Clustered Regularly Interspaced Short Palindromic Repeats

26. 4. Encapsulation of Dairy Starters
• Encapsulation of the dairy starters with effective base
materials can prevent the cultures from phage attack.
• Calcium alginate are commonly used to preparing
encapsulated beads for starter preservation

27. 4. Dairy plant and Cleanliness
• Phage development in these growth niches is controlled
by effective sanitation.
• Phages are disseminated throughout the dairy plant by
aerosol and human carriers.
• When preparing bulk starter, air drawn into the tank
when the culture medium cools should be filter
sterilized. Milk in cheese vats is most susceptible to
phage contamination during ripening and setting, so
these processes should be accomplished in closed
systems.

28. • Air entering cheese manufacturing rooms should be
under positive pressure of high-efficiency particulate air
(HEPA) filtered air.
• Whey should be removed to a physically separate facility,
because whey processing produces aerosols that can
carry phage particles.
• Plant personnel with exposure to whey should not be
allowed access to the milk-ripening or bulk starter
facilities.