REVIEWS

BACTERIOCINS: DEVELOPING
INNATE IMMUNITY FOR FOOD
Paul D. Cotter*, Colin Hill* and R. Paul Ross‡
Abstract | Bacteriocins are bacterially produced antimicrobial peptides with narrow or broad host
ranges. Many bacteriocins are produced by food-grade lactic acid bacteria, a phenomenon
which offers food scientists the possibility of directing or preventing the development of specific
bacterial species in food. This can be particularly useful in preservation or food safety
applications, but also has implications for the development of desirable flora in fermented food.
In this sense, bacteriocins can be used to confer a rudimentary form of innate immunity to
foodstuffs, helping processors extend their control over the food flora long after manufacture.
F O O D M I C RO B I O LO G Y

BACTERIOCINS
Bacterially produced, small,
heat-stable peptides that are
active against other bacteria and
to which the producer has a
specific immunity mechanism.
Bacteriocins can have a narrow
or broad target spectrum.
Alimentary Pharmabiotic
Centre, *Microbiology
Department, University
College Cork, Cork, Ireland.
‡
Moorepark Biotechnology
Centre, Teagasc, Cork,
Ireland.
Correspondence to C.H.
e-mail: c.hill@ucc.ie
doi:10.1038/nrmicro1240
Perhaps the oldest and most widespread antimicrobial
strategy in living systems is the use of antimicrobial
peptides by the innate immune systems of many forms
of life, from insects to plants to humans. Antimicrobial
peptides can be fast acting and have broad-spectrum
activity, which diminishes the possibility of resis-
tance developing in target species. One example of
antimicrobial peptides are the defensins, such as the
α-defensins, which are produced by neutrophils in the
small intestine and which act to reinforce the physical
barriers of the mucosal surface. However, the pro-
duction of antimicrobial peptides is not confined to
multicellular organisms. BACTERIOCINS are ribosomally
synthesized antimicrobial peptides produced by one
bacterium that are active against other bacteria, either
in the same species (narrow spectrum), or across
genera (broad spectrum) and, as with host defence
peptides1, cell signalling mechanisms can also be
involved. Producer organisms are immune to their own
bacteriocin(s), a property that is mediated by specific
immunity proteins.
It has been suggested that between 30–99% of the
Bacteria and Archaea make at least one bacteriocin2,3,
although genome analyses should ultimately provide
a more definitive figure4,5. Many bacteriocins are
produced by food-grade lactic acid bacteria (LAB),
and this offers the possibility of manipulating food
microbial ecosystems in a deliberate fashion — for
example, by using bacteriocins to protect food against
contamination with, or prevent the growth of, specific
pathogenic bacteria. Alternatively, narrow-host-range
bacteriocins can be used to influence a microbial
population in a specific ecosystem towards a particu-
lar outcome in which a desirable strain predominates
(competitive exclusion). In this sense, bacteriocins can
potentially be used as a form of innate immunity in
food to augment physical, physiological (for example,
pH) and chemical preservatives, and to influence the
probable final population in complex food systems.
The first description of bacteriocin-mediated inhi-
bition was reported 80 years ago, when antagonism
between strains of Escherichia coli was first discovered6.
Originally called ‘colicins’, to reflect the original pro-
ducer organism, gene-encoded antimicrobial peptides
produced by bacteria are now referred to as ‘bacterio-
cins’. Although the deliberate use of bacteriocins as
preservatives in food was formally proposed in 1951
REF. 7, it is likely that mankind has benefited from
the serendipitous production of bacteriocins in food
in the 8,000 years since cheese and other fermented
foods were first manufactured. Many studies have
revealed that the LAB used in cheese manufacture to
convert lactose to lactic acid also produce bacteriocins
that can influence the composition of the complex
cheese microflora and potentially inhibit adventitious
spoilage or pathogenic bacteria. Importantly, in 1928

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Timeline | Milestones in the commercial development of nisin
Colicins first described6.
1925 1928
LAB bacteriocins
first described8.
LANTIBIOTIC
Lanthionine-containing
bacteriocins. Lanthionines are
formed when a dehydrated
serine or threonine is covalently
bridged (through the sulphur
atom) with a cysteine.
BACTERIOLYSINS
Large, heat-labile, lytic proteins
produced by bacteria.
LIPID II
Lipid II (Undecaprenyl-
pyrophosphate–
MurNAc(pentapeptide)–
GlcNAc) has an essential role in
peptidoglycan synthesis by
translocating the peptidoglycan
subunits across the cell
membrane. Lipid II is also the
target of vancomycin, the drug
of last resort in the treatment of
MRSA.
778 | O CTOBER 2005
Nisin first marketed European Union
Nisin discovered9. in England. approval11.
1933 1947 1953 1969 1983 1988
Nisin named10. Food and Agriculture Organization/ US Food and Drug
World Health Organization approval. Agency approval.
it was observed that certain lactococcal strains had an
inhibitory effect on the growth of other LAB8. A pro-
teinaceous antimicrobial was also described in 1933 in
New Zealand9 and, in 1947, the responsible bacteriocin
was named nisin (or group N inhibitory substance)10
TIMELINE. Nisin was first marketed in England in 1953
and has since been approved for use in over 48 coun-
tries. Notably, nisin was assessed to be safe for food
use by the Joint Food and Agriculture Organization/
World Heath Organization Expert Committee on Food
Additives in 1969. In 1983, this bacteriocin was added
to the European food additive list as number E234
REF. 11 and, in 1988, it was approved by the US Food
and Drug Agency (FDA) for use in pasteurized,
processed cheese spreads.
The successful development of nisin from an initial
biological observation through regulatory approval to
commercial application is a model that has stimulated
a marked resurgence in bacteriocin research in recent
years, but similar success with other bacteriocins has
yet to be repeated on the same scale. In spite of this
sobering fact, we remain convinced that bacteriocins
can be exploited in food in various imaginative and
commercially important applications. However, to
fully realize this potential, it is first necessary to under-
stand the biology of bacteriocins, and, in particular,
to elucidate their structure–function relationships,
production, immunity, regulation and mode of action.
Biology of bacteriocins
Bacteriocins can be defined as bacterially produced,
small, heat-stable peptides that are active against
other bacteria and to which the producer has a spe-
cific immunity mechanism. The bacteriocins that will
probably have the most immediate potential in food
applications will be those produced by food-grade
LAB, as they are more likely to meet with regula-
tory approval owing to their origin, and they can be
readily introduced into fermented foods without any
concentration or purification. Bacteriocins that are
produced by LAB can be broad or narrow spectrum,
but in general, activity is directed against low-GC
Gram-positive species. Activity against Gram-negative
bacteria has been shown, but usually only in situations
where the integrity of the outer membrane has been
compromised.
| VOLUME 3
© 2005 Nature Publishing Group
Bacteriocins are a heterogeneous group of peptides
and proteins and as many as five main classes of LAB
bacteriocins have been mooted12–14. We propose that
this classification scheme should be revised and sug-
gest such a revision in TABLE 1. The proposed revised
classification scheme divides the bacteriocins into two
distinct categories: the lanthionine-containing LANTI
BIOTICS (class I) and the non-lanthionine-containing
bacteriocins (class II), while moving the large, heat-
labile murein hydrolases (formerly class III bacterio-
cins) to a separate designation called ‘BACTERIOLYSINS’. It
has recently been proposed that circular LAB bacterio-
cins should be regarded as class V bacteriocins14. We
suggest that these should be included in the non-
lanthionine-containing class II category. Class IV
was a classification reserved for bacteriocins that
require non-proteinaceous moieties for activity, but
no members of this class have been convincingly
demonstrated as yet, and so we have not included
this class in the new proposal.
Class I bacteriocins (lantibiotics). The lantibiotics
(lanthionine-containing antibiotics) are small peptides
(19–38 amino acids in length) that possess the epony-
mous lanthionine or β-methyllanthionine residues
(FIG. 1). These unusual residues form covalent bridges
between amino acids, which results in internal ‘rings’
and gives lantibiotics their characteristic structural
features. Furthermore, lantibiotics can contain other
unusual residues that result from post-translational
modification, including the substitution of d-alanines
for l-serines15–17.
The lantibiotics can be divided on the basis of their
structure and mode of action. In general, the elon-
gated amphiphilic cationic lantibiotics (for example,
nisin) are active through the formation of pores,
leading to the dissipation of membrane potential and
the efflux of small metabolites from sensitive cells.
By contrast, the globular lantibiotics (for example,
mersacidin) were originally defined as those lantibi-
otics that act through enzyme inhibition18. However,
it has now been established that nisin possesses
both mechanisms of action and that others, such
as lacticin 3147, a two-peptide lantibiotic, function
through the cooperative activity of two lanthionine-
containing peptides. Their complex structures make
the lantibiotics a difficult group to subclassify; this
is illustrated by a recent suggestion that they can be
subdivided into 11 subgroups based on the sequences
of the unmodified pro-peptides19. Some lantibiotics
are active in the single nanomolar range; that is, their
activity is several orders of magnitude higher than that
displayed by the antimicrobial peptides encountered
in the innate immune system.
Although a precise mechanism of action has not
been elucidated for every lantibiotic, it is apparent
that a docking molecule or target is involved, which
has been established as LIPID II in several cases20–24. The
binding of nisin to lipid II facilitates a dual mechanism
of action involving the prevention of peptidoglycan
synthesis and pore formation20,21. Nisin seems to have
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Table 1 | Suggested classification scheme for bacteriocins

Classification* Remarks/suggestions Examples
Class I
Lanthionine-containing Includes both single- and two-peptide Single-peptide: nisin, mersacidin,
bacteriocins/lantibiotics lantibiotics; up to 11 subclasses have been lacticin 481; two-peptide: lacticin
proposed19 3147, cytolysin
Class II
Non-lanthionine-containing Heterogeneous class of small peptides; Class IIa: pediocin PA1,
bacteriocins includes pediocin-like (subclass a bacteriocins), leucocin A; class IIb:
two-peptide (subclass b bacteriocins), cyclic lactacin F; class IIc: enterocin
(subclass c; formerly class V), non-pediocin AS48, reuterin 6; class IId:
single linear peptides (subclass d) lactococcin A, divergicin A
Bacteriolysins
Non-bacteriocin lytic proteins‡ Large, heat-labile proteins, often murein hydrolases Lysostaphin, enterolysin A
* Class IV bacteriocins (bacteriocins with non-proteinaceious moieties) are not included as no members have been demonstrated.
‡
Suggested that these are no longer considered bacteriocins (see main text).

a modular structure in that several N-terminally
located residues, which are also present in other nisin-
like, epidermin-like and streptin-like lantibiotics, have
been shown to form a binding cage that is essential
for the binding of nisin to the pyrophosphate of lipid II
REF. 22 . The C-terminus and intervening hinge
region, which is responsible for the movement of the
two termini relative to one another, are essential for
pore-formation21,25. It is also thought that the indi-
vidual peptides of some two-peptide lantibiotics might
represent discrete receptor-binding and pore-forming
modules26. The combined activity of these modules
helps to explain the extraordinarily high activity of at
least some lantibiotics (FIG. 2).
The cinnamycin-like lantibiotics represent the only
other class I bacteriocins for which a binding site — the
membrane phospholipid phosphatidylethanolamine
— has been identified27,28.
It is pertinent to note that there are examples of
bacteriocins of Gram-positive bacteria that also act
as virulence factors. The enterococcal two-peptide
lantibiotic cytolysin exerts activity against a broad
spectrum of cell types, including a wide range of
Gram-positive bacteria, eukaryotic cells such as
human, bovine and horse erythrocytes, retinal cells,
polymorphonuclear leukocytes and human intestinal
epithelial cells, and is associated with acute, terminal
outcomes in human infection29. Furthermore, another
non-lanthionine-containing peptide haemolysin,
streptolysin S, is involved in invasive infection by
some group A streptococci30.
Class II bacteriocins (non-lanthionine-containing
bacteriocins). The more common non-lanthionine-
containing bacteriocins are also small (<10 kDa)
heat-stable peptides but, unlike lantibiotics, they are
not subject to extensive post-translational modifica-
tion. The majority of class II bacteriocins are active
(also in the nanomolar range) by inducing membrane
permeabilization and the subsequent leakage of mol-
ecules from target bacteria (FIG. 2). Several different
groupings have been suggested13,31,32, but their hetero-
geneous nature makes rational classification difficult.
Two types are common to all classification systems and
are retained in our proposed classification scheme: the
class IIa pediocin-like or Listeria-active and the class IIb
two-peptide bacteriocins.
Pediocin-like bacteriocins have a narrow spectrum
of activity but display a high specific activity against
the food pathogen Listeria monocytogenes33,34. These
bacteriocins range from 37 (leucocin A and mesente-
ricin Y105 REFS 35,36 to 48 residues (carnobacteriocin
B2; REF. 37) and possess one or two disulphide bridges.
Conservation between peptides is most evident in
the hydrophilic, cationic N-terminal ‘pediocin box’
region, which contains the amino-acid sequence motif
YGNGVXCXXXXVXV, (in which X is any amino
acid)38,39, and is thought to facilitate nonspecific bind-
ing to the target surface40,41. The C-terminal domains,
which are located after a hinge region, are less con-
served and are thought to determine the non-listerial
antimicrobial spectrum. The C-terminal domains
have been used as a basis for the formation of three
further subdivisions42. The indications that these ter-
mini represent distinct modules has been confirmed
by the generation of hybrid class IIa bacteriocins,
which confirmed that the target specificity is deter-
mined by the C-terminal domain42–44. A link has been
established between the expression of the mannose
permease of the phosphotransferase system EIItMan in
L. monocytogenes and sensitivity to class IIa bacterio-
cins. Mesentericin Y105- and leucocin A-resistant
mutants of L. monocytogenes display reduced expres-
sion of the mptACD genes that encode this perm-
ease45–48. Although a physical interaction between
a class IIa peptide and this mannose permease has
not been established, the sensitisation of Lactococcus
lactis strains as a consequence of the introduction of
the mpt operon49 is consistent with the theory that
this permease functions as a receptor for class IIa
bacteriocins.
The two-peptide bacteriocins require the combined
activity of both peptides with a mechanism of action that
again involves the dissipation of membrane potential,
the leakage of ions and/or a decrease in intracellular ATP
concentrations50. These peptides display very low, if any,

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a
Ser Pro Arg Ile Thr Ser Ile Ser Leu Cys Thr Pro Gly Cys Lys Thr Gly

Leader 23 amino acids

OH OH SH SH
OH OH

Dehydration of selected
serines and threonines

Ser Pro Arg Ile Dhb Dha Ile Dha Leu Cys Dhb Pro Gly Cys Lys Dhb Gly

SH SH

Lanthionine
formation

Dha
Cleavage Ile Leu
Pro Gly

Ser Pro Arg Ile Dhb Ala S Ala Abu S Ala Lys Abu Gly

Lanthionine β-methyl-
lanthionine S

Leu
b Nisin
Ala Met
Dha
Ile Leu Pro Gly Gly Gly Ala Abu S Ala Ser Ile His Val Dha Lys
Ile Dhb Ala S Ala Abu S Ala Lys Abu S Ala Asn Met Lys Abu S Ala His

Asn
Gly Asn Lys
c Lacticin 3147 Ala
Trp Gly S
Trp
Tyr Ala Abu His
Ala
Lacticin A1 Asp Trp Leu Glu
Met
Ala Ala Dhb Asn Dhb Phe D-Ala Leu Ala S Ala Abu S Ala

S
Lacticin A2 Asn
Thr Thr Thr Lys Arg Ala
2-ob Dhb Pro Ala Dhb Pro Ala Ile D-Ala Ile Leu D-Ala Ala Tyr Ile Ala S Ala Pro Abu S Ala Abu S Ala

Figure 1 | Lanthionine synthesis and lantibiotic structure. As shown in a, lanthionine residues are formed when an
enzymatically dehydrated serine (dehydroalanine, Dha) condenses with the sulphydryl group of a neighbouring cysteine (Cys).
This forms a bridge between the two residues, thereby creating a ring within the modified peptide or lantibiotic. When the
partners are threonine (Thr) and cysteine, the novel residue is a β-methyllanthionine. The resulting lanthionine and
β-methyllanthionine bridges are indicated in pink as Ala–S–Ala (alanine–S–alanine) and Abu–S–Ala (aminobutyrate–S–alanine),
respectively. Many lantibiotics also contain dehydrated serines (Ser) and threonines (dehydrobutyrine, Dhb). Lantibiotics can be
composed of a single peptide (nisin, panel b) or two peptides acting in synergy (lacticin 3147, panel c). The dehydration and
ring formation reaction can be catalysed by two enzymes (NisB and NisC in the case of nisin) or a single enzyme (LcnM in the
case of lacticin 481). Several additional modified residues can be found in lantibiotics, including lysinoalanine, lanthionine
sulphoxide, D-alanine, allo-isoleucine, erythro-3-hydroxyaspartate, 2-oxopyruvate, 2-oxobutyrate, hydroxypyruvate,
S-(2-aminovinyl)-D-cysteine and S-(2-aminovinyl)-3-methyl-D-cysteine.

bacteriocin activity when tested individually. Although
members of this subgroup are relatively heterogeneous,
it has been proposed that they could be subdivided into
type E (enhanced) and type S (synergistic) peptides51.
The class IIc (formerly class V) bacteriocins are
grouped on the basis that their N- and C-termini are
covalently linked, resulting in a cyclic structure52,53.
Although relatively few class IIc bacteriocins have been
identified, we propose two subdivisions designated
subclass c(i) (comprising enterocin AS48 and the
non-LAB circularin A) and subclass c(ii) (comprising
gassericin A, reutericin 6, the non-LAB butyrivibriocin
AR10 and, although a circular structure has not yet
been established, acidocin B) on the basis of percentage
amino-acid sequence identity52. The class IIc bacterio-
cins gassericin A and reutericin 6 are the only examples
of non-lantibiotic LAB bacteriocins that contain
d-amino acids54.

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Class II Class I Bacteriolysins
(Sakacin) (Nisin) (Lysostaphin)
Cell wall
Lipid II
Cell membrane
Peptidoglycan subunit
Figure 2 | Mode of action of lactic acid bacteria bacteriocins. Lactic acid bacteria (LAB)
bacteriocins can be grouped on the basis of structure, but also on the basis of mode of action.
Some members of the class I (or lantibiotic) bacteriocins, such as nisin, have been shown to have
a dual mode of action. They can bind to lipid II, the main transporter of peptidoglycan subunits
from the cytoplasm to the cell wall, and therefore prevent correct cell wall synthesis, leading to
cell death. Furthermore, they can use lipid II as a docking molecule to initiate a process of
membrane insertion and pore formation that leads to rapid cell death. A two-peptide lantibiotic,
such as lacticin 3147, can have these dual activities distributed across two peptides, whereas
mersacidin has only the lipid-II-binding activity, but does not form pores. In general, the class II
peptides have an amphiphilic helical structure, which allows them to insert into the membrane of
the target cell, leading to depolarisation and death. Large bacteriolytic proteins (here called
bacteriolysins, formerly class III bacteriocins), such as lysostaphin, can function directly on the cell
wall of Gram-positive targets, leading to death and lysis of the target cell.
The remaining bacteriocins are usually combined in
a ‘miscellaneous’ or ‘one-peptide non-pediocin linear’
group (class IId). In some instances, these have been
further subdivided on the basis of leader sequences31.
Non-bacteriocin lytic proteins (bacteriolysins).
Bacteriolysins (formerly class III bacteriocins) are
large, heat-labile antimicrobial proteins. They have a
domain-type structure, in which different domains have
functions for translocation, receptor binding, and lethal
activity. Only four LAB bacteriolysins55–59 have been
genetically characterized so far, although other non-
LAB bacteriolysins that are of interest to food micro-
biologists have been identified51,60,61. Their mechanism
of action is distinct from that of bacteriocins as they
BACTERIOCINOGENIC
function through the lysis of sensitive cells by catalys-
Bacteriocin-producing strains ing cell-wall hydrolysis (FIG. 2). These proteins are also
are said to be bacteriocinogenic. modular in structure and have a catalytic domain at
NATURE REVIEWS | MICROBIOLOGY
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REVIEWS
the N-terminus that shows homology to endopepti-
dases, and a C-terminus that probably represents the
target recognition site62,63. Unlike the ‘true’ bacteriocins,
they do not always have specific immunity genes that
accompany bacteriocin structural genes, but might rely
on modifications of the producer cell wall to impart
resistance (see below).
Molecular biology of bacteriocins
The widespread phenomenon of bacteriocin production
among LAB is undoubtedly partly due to the fact that
the relevant genes are often associated with transferable
elements such as conjugative transposons or plasmids.
This natural association can be exploited to facilitate
heterologous bacteriocin production. For example, the
10 genes that are required for the production of, and
immunity to, the two-peptide lantibiotic lacticin 3147
are located on a 60-kb conjugative plasmid, which has
allowed this trait to be transferred in a non-recombinant
manner from the parent strain L. lactis DPC3147 to a
wide variety of commercial starter strains, by simply
selecting for bacteriocin immunity as a marker for
plasmid transfer64. Bacteriocins are usually synthesized
as an inactive pre-peptide that includes an N-terminal
leader sequence. The leader sequence presumably
maintains the bacteriocin in an inactive form within
the producer cell, facilitates interaction with the trans-
porter and, probably in the case of lantibiotics, has a
role in recognition by the modification machinery65–68.
This leader is usually cleaved during export by a dedi-
cated bacteriocin-transport system or, less frequently,
by the general secretion (Sec) pathway of the cell. It is
notable that a few bacteriocins that seem to lack leader
sequences have also been identified31. The genes that
encode the structural pre-peptides are usually closely
associated with genes that encode products involved in
regulation, export, self-immunity and — in the case of
the lantibiotics — modification.
The export of bacteriocins is usually achieved by a
dedicated membrane-associated ATP-binding cassette
(ABC) transporter that can also contain a proteolytic
N-terminal domain belonging to the family of cysteine
proteases that is responsible for cleavage of the leader
peptide69,70. In the case of some lantibiotics, cleavage
might be due to a specific serine protease71. For class II
peptides, accessory proteins are thought to facilitate
membrane translocation and/or leader peptide cleav-
age, but their specific role has yet to be established. It
should be noted that there are several variations to this
general scheme, which involve both N- and C-terminal72
or extracellular cleavage73,74.
Regulation of bacteriocin production and immunity
is most frequently mediated through two-component
signal-transduction systems, often as part of a quorum-
sensing mechanism (FIG. 3). In certain instances, more
than one response regulator can be involved75,76. Other
types of regulators, such as repressors of the Xre family,
have also been described77,78.
BACTERIOCINOGENIC bacteria also have genes that
encode immunity mechanisms. These systems enable
a distinction between ‘self ’ (producer) and ‘non-self ’
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Enterocin A Enterocin F
EntD
EntK
EntT
EntR
+
Pent
0 1 2 3 5 6 7 8 kb
ent A I F K R T D
Figure 3 | Regulation by quorum sensing. In several instances it has been established that
bacteriocin production is regulated by quorum sensing. In the case of the lantibiotics nisin and
subtilin, the structural peptide itself functions as a pheromone to induce its own production to
high levels once a cell-density-dependent autoinduction loop is activated163–165. Although there
is an example of a class II bacteriocin acting as a pheromone144, the presence of another
induction peptide, which often shows many of the physico-chemical properties of bacteriocins
and can function at concentrations at low as 10–17 M REF. 166, is more common. As illustrated
in the figure, the regulation of enterocin A production involves one such three-component
system. The genes that encode the structural protein enterocin A (EntA) and the induction
factor enterocin F (EntF) are co-transcribed from the Pent promoter and these proteins are
transported by a putative ATP-binding cassette transporter (EntT) through a mechanism that
might involve the accessory protein (EntD). As with other bacteriocins, the other two-
component system involved is a membrane-bound histidine kinase (EntK), which detects
extracellular EntF, and a cytoplasmically located response regulator (EntR) that mediates a
response — induction of transcription from the Pent promoter, which leads to increased
bacteriocin production. In some cases, additional factors such as co-culture with potential
target cells167–169 or even gastric transit170 can be required for successful induction. An
understanding of quorum sensing and inducible bacteriocin production has facilitated the
development of systems that permit inducible overexpression of desirable proteins164,171,172.
(for example, food spoilage or pathogenic target
bacteria). Two distinct systems are involved in class I
and class II immunity, although there are some excep-
tions79. Protection can be provided by a dedicated
immunity protein and/or a specialised ABC-trans-
porter system involving two or three subunits that
probably pumps the bacteriocin from the producer
membrane76,80–83. For class I bacteriocins, immunity is
provided by one or both of these systems82, for class
IIa, IIb and IId bacteriocins, immunity is provided by
an immunity protein alone, and class IIc bacteriocins
rely on an ABC-transporter system84. Although immu-
nity proteins rarely resemble one another, the general
mechanism through which they function is prob-
ably similar, involving either the sequestration of the
structural protein or an antagonistic competition for
a receptor85,86. Such immunity mechanisms are highly
specific and usually do not provide protection against
other bacteriocins82,87,88, although again, there are some
exceptions39,89,90. It has been shown that immunity to
bacteriolysins is brought about through the alteration
of interpeptide cross-bridges in peptidoglycan91.
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Resistance to bacteriocins
The potential for the development of resistance
among target species is an obvious cause for concern
if bacterio cins are to be used extensively in food
preservation strategies. Most research in this area
has focused on specific bacteriocins, such as nisin
and some of the class IIa peptides. The frequency of
spontaneous resistance to nisin in L. monocytogenes
varies from 10–2 to 10–7 in a strain-dependent manner92,
whereas stable, nisin-resistant mutants of Streptococcus
pneumoniae can arise (MIC increases from 0.4 to
6.4 mg l–1) following serial exposure to the lantibiotic93.
In these spontaneous mutants, resistance correlates
with cell envelope changes such as alterations in mem-
brane charge and fluidity94,95, cell wall thickness96, cell
wall charge97–99 and combinations thereof 100, which
arise after direct exposure to a low level of lantibiotic
or as part of an adaptive response to another stress101.
The specific mechanism(s) by which cells become
resistant to nisin is not well understood although it is
apparent that variations in the lipid II content are not
responsible102. Genetic loci that are associated with the
development of enhanced nisin resistance103–105, or an
innate tolerance of nisin, have been identified98,106,107.
In the latter example, cell envelope charge seems to be
the most important consideration.
The most well-studied mechanism of resistance
to class II bacteriocins is that observed in class IIa-
resistant L. monocytogenes, in which resistance
seems to be linked to reduced expression of a man-
nose permease of the phosphotransferase system.
Similar to nisin-resistant mutants, other factors such
as cell membrane fluidity108 and cell surface charge109
impact on resistance. With further research, additional
mechanisms will undoubtedly become apparent. The
production of a bacteriocin by a Streptococcus mutans
strain is inhibited by Streptococcus gordonii through
the inactivation of the corresponding induction
peptide, which provides an interesting example of
bacteriocin-mediated communication that involves
competing species110.
Further studies will be required to determine the
frequency with which resistance to other bacteriocins
occurs. The risk of the development of large-scale,
industrially significant resistance might best be lim-
ited by the intelligent use of hurdle technology, that
is, the concept of combining several factors (including
pH, osmolarity and temperature) to preserve food.
Although the combined use of different bacteriocins
can be successful, cross-resistance to class I and class II
bacteriocins has been found in some instances100,104,111.
It is worth noting that bacteriocins used correctly in
a hurdle approach should only have to control low
levels of contaminating organisms, as they should
be used only to augment, and not replace, good
food manufacturing practice. This creates a different
situation to the use of antibiotics in clinical settings,
where these inhibitors are used to eliminate estab-
lished infections and usually high numbers of target
bacteria, thereby greatly increasing the probability
of resistance development.
www.nature.com/reviews/micro

GRAS
An acronym for substances that
are ‘generally recognized as safe’.
This group includes several
hundred substances that experts
consider safe for use in food on
the basis of either a history of
safe use before 1958 or on
published scientific evidence.
NATURE REVIEWS | MICROBIOLOGY
Recent developments in bacteriocin biology
Many exciting developments have contributed to our
understanding of bacteriocin biology and have high-
lighted innovative ways in which these peptides can
be used; for example, cell-signalling systems involved
in the induction of bacteriocin production have been
investigated and used as biological tools (FIG. 3).
One of the most significant developments has been
the discovery that the activity of nisin is mediated by
the binding of lipid II REFS 20,21. Whereas lipid II
is also the target for therapeutic antibiotics such as
vancomycin, it has been established that a set of core
amino acids at the N-terminal domain of nisin bind
to an invariable region of lipid II REFS 22,112, thereby
militating against resistance like that observed in
vancomycin-resistant enterococci (VRE). These
results indicate that nisin could act as a template
for the design of novel drugs that bind an essential,
invariable antimicrobial target. Although the defini-
tive role of EIItMan as the target or docking molecule
for class IIa bacteriocins has yet to be established, an
ever-improving appreciation of the modular nature of
these bacteriocins will facilitate the creation of hybrid
proteins with different antimicrobial spectra as a
consequence of C-terminal variation42. Attempts to
create bioengineered derivatives of bacteriocins have
met with some rare, but notable, successes113–116. The
absence of post-translational modifications in class II
bacteriocins makes them more amenable to change,
a fact that is best illustrated by the creation of hybrid
bacteriocins43,44. Whereas bioengineered peptides
are usually expressed from a derivative of the origi-
nal bacteriocin-producing strain, it has been shown
that class II peptides can be produced heterologously
by Gram-negative bacteria or yeast117,118. Although
the heterologous production of a fully modified
lantibiotic by non-Gram-positive microorganisms
has yet to be achieved, it has recently been shown
that purified modification enzymes can be used to
introduce lanthionines into synthetic peptide sub-
strates68. We predict that by combining novel insights
into structure–function relationships with enhanced
mutagenesis and production technologies, the bio-
engineering of bacteriocins will enter a new era in
which the identification of derivatives with enhanced
activity will be a more frequent event.
One should note, however, that although some of
this newly generated knowledge will undoubtedly result
in novel and exciting applications, a significant suite of
applications have already been demonstrated, even if,
in many instances, only at the laboratory scale.
Industrial applications
Food systems. Food processors face a major challenge
in an environment in which consumers demand safe
foods with a long shelf-life, but also express a prefer-
ence for minimally processed products that do not
contain chemical preservatives. Bacteriocins are an
attractive option that could provide at least part of the
solution. They are produced by food-grade organisms,
they are usually heat stable and they can inhibit many
© 2005 Nature Publishing Group
REVIEWS
of the primary pathogenic and spoilage organisms that
cause problems in minimally processed foodstuffs.
However, at present, only nisin and pediocin PA1/AcH
have found widespread use in food. The form of nisin
used most widely in food is Nisaplin (Danisco), which
is a preparation that contains 2.5% nisin with NaCl
(77.5%) and non-fat dried milk (12% protein and 6%
carbohydrate). The use of pediocin PA1 for food bio-
preservation has also been commercially exploited in
the form of ALTA 2431 (Quest), which is based on LAB
fermentates generated from a pediocin PA1-producing
strain of Pediococcus acidilactici119. Its use is covered by
several US and European patents119,120.
When screening for a bacteriocin with a food appli-
cation in mind, there are several important criteria:
first, the producing strain should preferably have ‘gen-
erally recognized as safe’ (GRAS) status; and second, the
bacteriocin should have a broad spectrum of inhibition
that includes pathogens, or have activity against a par-
ticular pathogen. Third, the bacteriocin should be heat
stable; fourth, have no associated health risks; fifth, its
inclusion in products should lead to beneficial effects
such as improved safety, quality and flavour; and sixth,
it should have high specific activity121. Bacteriocins have
been shown to have potential in the biopreservation
of meat, dairy products, canned food, fish, alcoholic
beverages, salads, egg products, high-moisture bakery
products, and fermented vegetables, either alone, in
combination with other methods of preservation, or
through their incorporation into packaging film/food
surfaces (for comprehensive reviews, see REFS 122124).
Bacteriocins can be introduced into food in at least
three different ways: in fermented food, bacteriocins
can be produced in situ by bacterial cultures that sub-
stitute for all or part of the starter culture; purified or
semi-purified bacteriocins (for example, Nisaplin) can
be added directly to food; or an ingredient based on
a fermentate of a bacteriocin-producing strain can be
used (for example, ALTA 2431). Although bacterio-
cins with a wide spectrum of activity are usually the
most sought after, other factors including pH optima,
solubility and stability are as important and are major
considerations in choosing a particular inhibitor for a
particular food or target bacterium. Furthermore, the
antimicrobial spectra of a variety of LAB bacteriocins
can be extended to encompass Gram-negative bacteria
through their use in combination with measures that
affect the integrity of the outer membrane, such as tem-
perature shock, high pressure, chelators and eukaryotic
antimicrobial peptides125–133. There are also rare natu-
ral (for example, AS48 REF. 134) and bioengineered
bacteriocins114 that possess inherent activity against
Gram-negative microorganisms.
There is also a niche for narrow-spectrum bac-
teriocins. This is true of fermented foods, in which
contamination with L. monocytogenes is a problem.
L. monocytogenes is the cause of approximately 25%
of deaths caused by food-borne pathogens in the US
annually135 and, as a consequence of the zero-tolerance
standards for the organism in ready-to-eat (RTE) food,
was responsible for 71% of all recalls of food products
VOLUME 3 | O CTOBER 2005 | 783
REVIEWS
owing to bacterial contamination in the US between
1993 and 1998 REF. 136. In mid-2003, the Food Safety
and Inspection Service (FSIS) announced a ruling that
required the manufacturers of RTE food to take further
steps to address the problem posed by the presence of
L. monocytogenes in consumables. This ruling encour-
ages the implementation of technologies that can kill
the bacteria or prevent its growth after cooking or
packaging. The Listeria-active class IIa bacteriocins
seem to provide an ideal solution for such applica-
tions, especially with respect to fermented foods, as
any potential L. monocytogenes contamination can be
negated without impacting on the success of the fer-
mentation itself owing to the low activity of class IIa
bacteriocins against lactococci39.
Bacteriocins can also be used to promote quality,
rather than simply to prevent spoilage or safety prob-
lems. For example, bacteriocins can be used to control
adventitious non-starter flora such as non-starter
lactic acid bacteria (NSLAB) in cheese and wine.
The uncontrolled growth of NSLAB can cause major
economic losses owing to calcium-d-lactate form-
ation137 and slit defects in cheeses, and the production
of detrimental compounds in wine. Bacteriocin-
producing starters and adjuncts (one- or two-strain
strategies) have been found to significantly reduce
these problems138–142. However, as some NSLAB such
as lactobacilli and other starter adjuncts in cheese, and
Leuconostoc oenos and Pediococcus damnosus in some
red wines can improve flavour, the complete elimina-
tion of NSLAB is not always desirable143. This problem
has been overcome through the use of a three-strain
system in which an adjunct strain with reduced bac-
teriocin sensitivity (obtained on repeated exposure to
increasing concentrations of the bacteriocin) is used
with a bacteriocin-producing starter144 (FIG. 4).
Bacteriocins can also be applied in other ways to
enhance food fermentation. This has been shown dur-
ing semi-hard and hard cheese manufacture in which
bacteriocin production brings about the controlled
lysis of starter LAB, which results in the release of intra-
cellular enzymes and ultimately accelerated ripening
and even improved flavour139,140,145,146.
Although traditionally, the use of bacteriocins is
associated with the preservation of food, in the near
future food might merely act as a vehicle for the deliv-
ery of bacteriocin-producing probiotic bacteria. The
production of antimicrobials by a probiotic culture is
a desirable trait as they are thought to contribute to
the inhibition of pathogenic bacteria in the gut147–149.
Whereas bacteriocins in food are degraded by the
proteolytic enzymes of the stomach, probiotic bacteria
might be ingested in a form that facilitates gastric tran-
sit, allowing the in vivo production of the bacteriocin
in the small or large intestine. It has also been specu-
lated that recombinant probiotic strains that can be
induced to produce bacteriolysin could be developed
to facilitate the in vivo delivery of bioactive compounds
that are produced intracellularly150.
Unlike most other preservation methods such as
heat or low pH, which are essentially indiscriminate in
784 | O CTOBER 2005 | VOLUME 3
© 2005 Nature Publishing Group
their antimicrobial effect, it is this ability to precisely
influence the developing flora in an otherwise perish-
able food that led us to describe the use of bacteriocins
as a form of ‘innate immunity’ for food. As already
described, the inclusion of Listeria-active class IIa
bacteriocins can specifically prevent the growth of this
pathogen, without affecting harmless LAB, or bacterio-
cin-tolerant strains can be introduced into an otherwise
hostile food environment. It is unlikely that the use of
bacteriocins in food will negatively impact on the natu-
ral flora of either the human (or animal) host, or on
the environment. The low level of bacteriocins required
to eliminate or reduce small numbers of pathogenic
or spoilage organisms in food are unlikely to have an
impact on more microorganism-rich environments. In
any event, bacteriocins are unlikely to survive gastric
transit, as they are sensitive to proteolytic degradation.
Clinical applications. Although this review has
emphasized the role (existing and potential) of LAB
bacteriocins in food, the non-toxicity of lantibiotics
and their activity against Gram-positive human and
animal pathogens has led to research investigating
their potential clinical application. In particular, the
elucidation of the precise mechanism of action of
some lantibiotics and their activity against multidrug-
resistant pathogens by a novel mechanism makes them
an attractive option as possible therapeutic agents. The
broad-spectrum lantibiotics could theoretically be of
use against any clinical Gram-positive human or ani-
mal pathogen. For example, the two-peptide lantibiotic
lacticin 3147 has in vitro activity against Staphylococcus
aureus (including methicillin-resistant S. aureus
(MRSA)), enterococci (including VRE), streptococci
(S. pneumoniae, Streptococcus pyogenes, Streptococcus
agalactiae, Streptococcus dysgalactiae, Streptococcus
uberis, Streptococcus mutans), Clostridium botulinum,
and Propionibacterium acnes151. Initial in vivo trials
with animal models have demonstrated the success
of lantibiotics in treating infections caused by S. pneu-
moniae152, and MRSA153,154, and in preventing tooth
decay and gingivitis155–159. The use of nisin for human
clinical applications has been licensed to Biosynexus
Incorporated by Nutrition 21 and ImmuCell
Corporation has licensed the use of the anti-mastitic
nisin-containing product Mast Out to Pfizer Animal
Health. Bovine mastitis is defined as an inflammation
of the udder and is the most persistent disease in dairy
cows. Nisin is also used as an active agent in Wipe-
Out (a teat wipe), and lacticin-3147-containing Teat
Seals (Cross Vetpharm Group Ltd) have been shown to
prevent deliberate infection by mastitic staphylococci
and streptococci in animal challenge trials159 (FIG. 4).
A strain that produces the lantibiotic mutacin 1140 is
entering Phase I clinical trials in the US with a view
to replacement therapy, and the dietary supplement
BLIS K12 throat guard, which contains a Streptococcus
salivarius that produces two lantibiotics salivaricin A2
and B, is sold in New Zealand as an inhibitor of the
bacteria responsible for bad breath160. From a non-
antimicrobial medical perspective, the cinnamycin-like
www.nature.com/reviews/micro
 REVIEWS

a Food quality Bac– Bac+

b Food safety c Veterinary medicine

104 80,000
Bac–
L. monocytogenes

S. aureus
103 20,000

102 10,000
Bacteriocins
Bac+
nd
1 2 3 4 5 Teat seal Teat seal and
Number of days lacticin 3147

d Human medicine Asn Ala S CH

Dha
Trp Leu Pro Gly Phe Tyr
CH
Phe Lys Ala S Ala Abu S Ala Ala Arg Dhb Gly Ala S Ala
N
H
Figure 4 | Selected applications of bacteriocins. a | Food quality. A cheese made with a commercial starter culture (Bac–) will
develop an undefined flora called non-starter lactic acid bacteria (typified by different fingerprints generated by random amplified
polymorphic DNA patterns). However, a cheese inoculated with the same commercial strain that can produce a bacteriocin
(Bac+) (lacticin 3147, in this example) and a resistant adjunct strain of Lactobacillus, chosen for a flavour attribute, will develop a
single defined culture once the starter culture has died off, offering the cheese manufacturer control over previously adventitious
flora development. b | Food safety. A simple example of the role of bacteriocins in food safety is the production of cottage
cheese with a starter culture that produces a bacteriocin with activity against Listeria monocytogenes, which results in a cheese
that is inherently anti-Listeria. c | Veterinary medicine. A teat seal is a physical barrier against infection. Here, a bacteriocin was
incorporated into the teat seal and the teat was challenged with Staphylococcus aureus. The number of staphylococci
recovered from 14 teats with or without bacteriocin is shown. d | Human medicine. A Streptococcus mutans strain that cannot
produce acid, but that produces the lantibiotic mutacin (shown), can competitively exclude acidogenic S. mutans, thereby
offering protection against tooth decay173.

lantibiotics have attracted interest owing to their novel
activities against the functions of medically important
specific human enzymes, such as phospholipase A2
and angiotensin-converting enzyme, and nisin has also
been found to have contraceptive efficacy161,162.
Conclusions
For the past 50 years, bacteriocins have frequently
been proposed as the solution to a myriad of food-
contamination problems, but nisin and, to a lesser
extent, pediocin PA1, remain the only examples of
the large-scale deliberate use of these peptides in food
applications. The under-utilization of bacteriocins in
food can perhaps be ascribed to a combined lack of
awareness of what bacteriocins can achieve in food
systems and a lack of enthusiasm to move away from
existing food-preservation techniques. However,
although there have been several ‘false dawns’ herald-
ing the bacteriocin revolution, modern consumer and
regulatory demands associated with minimal pro-
cessing might now provide an opportunity for their
more widespread application. Bacteriocin screening
programmes have yielded a large arsenal of bacterio-
cins with different properties, target species and
producer organisms. Furthermore, if even the most
conservative of estimates concerning the percentage of
bacteria that produce bacteriocins are confirmed, then
many other bacteriocins remain to be identified and
exploited. Whether deliberately added or produced
in situ by food bacteria, bacteriocins can play a benefi-
cial role in the control of undesirable flora and in the
establishment of desirable microbial populations. It is
this ability of bacteriocins to direct microbial flora that
can be described as a type of programmable innate
immunity for food.
Genomic advances will also contribute to the identi-
fication of novel bacteriocins, and enhanced under-
standing of regulatory mechanisms will allow us to
determine how to switch on production when desired
and even how to generate overproducing strains. By
determining the specific activity of bacteriocins
against particular spoilage/pathogenic bacteria, ensur-
ing stability and assessing the likelihood of emerging
resistance, it should be possible to pre-determine

NATURE REVIEWS | MICROBIOLOGY VOLUME 3 | O CTOBER 2005 | 785

© 2005 Nature Publishing Group
REVIEWS

which bacteriocin is best suited to a particular food
application. Such an application has been exemplified
by the tailored use of class IIa bacteriocins in the treat-
ment of L. monocytogenes. From a clinical perspective,
the emergence of drug-resistant pathogens makes
the identification of novel antimicrobials even more
important. Indeed, the adoption of protein-engineer-
ing strategies using existing bacteriocin structures as a
blueprint provides an attractive approach to tailoring
the biological activity and spectrum of inhibition of
bacteriocins. Given the enormous challenges faced
by both the food and pharmaceutical industries, the
biggest question might not be whether bacteriocins
will be used more extensively, but rather which of
these fields will more passionately embrace their use.
We believe that the food industry is well positioned,
given the exacting demands for safe and natural foods
imposed by consumers, to use the arsenal of bacterio-
cins produced by food-grade bacteria to develop foods
with innate immunity that can influence complex,
adventitious microbial populations in a predictable
and beneficial manner.

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Acknowledgements
The authors are supported by the Irish Government under the
National Development Plan (2000–2006) and by Science
Foundation Ireland.
Competing interests statement
The authors declare no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez: http://www.ncbi.nlm.nih.gov/Entrez
Escherichia coli | Lactococcus lactis | Leuconostoc oenos |
Listeria monocytogenes | Staphylococcus aureus |
Streptococcus gordonii | Streptococcus mutans |
Streptococcus pneumoniae
FURTHER INFORMATION
Alimentary Pharmabiotic Centre:
http://apc.ucc.ie/content/bacteriocins.htm
Gateway to Government Food Safety Information:
http://www.foodsafety.gov/~dms/lmr2-toc.html
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