Download Resistance and Adaptation to Food Antimicrobials

Scientific Status Summary
Resistance and Adaptation
to Food Antimicrobials,
Sanitizers, and Other
Process Controls
This IFT Scientific Status Summary discusses the potential for
microorganisms to become resistant to antimicrobials and
sanitizers used in food processing.
P. Michael Davidson and Mark A. Harrison
M
ost antimicrobials used in food manufacture have been in use
for about 50 to 100 years. A few antimicrobials, e.g., sulfites and
nitrites, have been in use for an even longer period of time.
Similarly, sanitizing agents, used to reduce microorganisms on processing equipment, have been in use for nearly 100 years. Concern has been
recently raised, however, about the potential for target pathogenic
Author Davidson is Professor, Dept. of Food Science and
Technology, University of Tennessee, 2509 River Dr.,
Knoxville, TN 37996-4539. Author Harrison is Professor,
Dept. of Food Science and Technology, University of
Georgia, Athens, GA 30602. Both authors are Professional
Members of IFT.
VOL. 56, NO. 11 • NOVEMBER 2002
microorganisms to develop resistance to these compounds.
Surprisingly, despite the considerable length of time that food antimicrobials and equipment sanitizers have been used in the food industry,
there is little data about the development of microbial resistance to these
compounds. This lack of data might be viewed as a good indication that
resistance development is probably not a major problem.
Concern remains, however, for three reasons. One concern is the increasing incidence of microorganisms exhibiting resistance to antibiotics
used for therapeutic purposes in human and animal medicine. A second
concern is the increasing reliance on antimicrobials and sanitizers as primary tools for controlling the outgrowth of pathogens in foods. A third
concern is the evidence indicating that tolerance to antimicrobials, sanitizers, and other preservation processes may be generated within microorganisms exposed to certain stresses.
If antimicrobials and sanitizers are to play a major role in effective control of foodborne pathogens, food manufacturers and others within the
food industry must know more about the potential for development of resistance among target microorganisms. This Scientific Status Summary explores the potential for such resistance development. In addition, the
Summary examines the interrelationship of resistance to antimicrobials
FOODTECHNOLOGY 69
Scientific Status Summary
United States are listed in Table 1.
Naturally occurring antimicrobials
include compounds that originate from
microbial, plant and animal sources. A
subgroup of naturally occurring antimicrobials is the bacteriocins, proteins
produced by lactic acid bacteria, e.g.,
Lactococcus, Lactobacillus, and Pedicoccus species, and a few other bacteria.
Only a few naturally occurring antimicrobials, such as nisin, natamycin,
lactoferrin and lysozyme, have regulatory approval for application to foods
(Table 1). Many additional antimicrobials, especially those derived from
microorganisms, hold the potential for
Food Antimicrobials and Sanitizers
regulatory approval in the future (Table
Food antimicrobials are compounds
2).
used to extend the lag phase or kill miFood antimicrobials are
traditionally used for their
ability to inhibit spoilage miTable 1—Traditional and naturally occurring food antimicrobials approved by the
croorganisms and, thus, proFood and Drug Administration. From Davidson and Branen (1993) and CFR (2001)
long shelf life and preserve
Compound(s)
Microbial target
Primary food applications Title 21 CFR designationa
food quality. Recently, however, antimicrobials have
been used increasingly as priAcetic acid, acetates,
Yeasts, bacteria
Baked goods, condiments,
184.1005, 182.6197,
diacetates, dehydroacetic
confections, dairy products,
184.1754, 184.1185,
mary interventions to inactiacid
fats/oils, meats, sauces
184.1721, 172.130
vate or inhibit the outgrowth
of pathogenic microorganBenzoic acid, benzoates
Yeasts, molds
Beverages, fruit products,
184.1021, 184.1733
isms in foods. Although food
margarine
antimicrobials have been
used for many years, few of
Dimethyl dicarbonate
Yeasts
Beverages
172.133
these substances are used exLactic acid, lactates
Bacteria
Meats, fermented foods
184.1061, 184.1207,
clusively to control the
184.1639, 184.1768
growth of specific foodborne
pathogens. Examples of
b
Lactoferrin
Bacteria
Meats
—
those used exclusively to
control specific pathogens
Lysozyme
Clostridium botulinum,
Cheese, frankfurters,
184.1550c
other bacteria
cooked meat and poultry
are nitrite to inhibit the
products
growth of Clostridium botulinum in cured meats, selected
Natamycin
Molds
Cheese
172.155
organic acid sprays to reduce
pathogens on beef carcass
Clostridium botulinum,
Cheese, other products
184.1538d
Nisin
surfaces, nisin and lysozyme
other bacteria
to inhibit growth of C. botuClostridium botulinum
Cured meats
172.160, 172.170,
Nitrite, nitrate
linum in pasteurized process
172.175, 172.177
cheese, and lactate and diacetate to inactivate Listeria
Parabens (alkyl esters
Yeasts, molds,
Beverages, baked goods,
184.1490, 184.1670,
monocytogenes in processed
(propyl, methyl, heptyl) of
bacteria (Gramsyrups
172.145
meats (FDA, 2000). Generalp-hydroxybenzoic acid)
positive)
ly, these compounds serve as
Propionic acid,
Molds
Bakery products, dairy
184.1081, 184.1221,
the primary microbial conpropionates
products
184.1784
trols among a combination
of inhibitors and inhibitory
Sorbic acid, sorbates
Yeasts, molds, bacteria
Most foods, beverages,
182.3089, 182.3225,
conditions (e.g., low pH and
wines
182.3640, 182.3795
low temperature). Such use
of combinations of several
Sulfites
Yeasts, molds
Fruits, fruit products,
Various
potato products, wines
microbial controls (multiple
a
interventions) is sometimes
These are the Food and Drug Administration’s designations in Title 21 of the Code of Federal Regulations. Food antimicrobials approved by the U.S.
Dept. of Agriculture’s Food Safety and Inspection Service for use in meat products are listed in Sections 424.21 and 424.22 of Title 9 of the CFR.
called “hurdle technology”
b
FDA/CFSAN (2001a)
(Leistner, 2000; Leistner and
c
FDA/
CFSAN (2001b)
d
Gorris, 1995).
FDA/CFSAN (2001c)
with resistance to environmental controls (e.g., sanitizers) used within food
manufacturing.
This Summary specifically addresses:
(1) antimicrobial and sanitizer functions, (2) mechanisms for the development of resistance and tolerance to antimicrobials and sanitizers, (3) results of
studies on resistance development to
traditional and novel food antimicrobials and the effect of microbial adaptation and tolerance to these substances,
and (4) impact of microbial resistance
development on food safety.
70
FOODTECHNOLOGY
croorganisms. They are different than
therapeutic antibiotics (e.g., penicillin,
tetracyclines) used to treat human or
animal disease. Food antimicrobials are
sometimes called “preservatives.” The
term “preservative,” however, often includes antioxidants in addition to antimicrobials. “Antimicrobial” is the more
specific term in the context of this discussion and, therefore, is used throughout this document. Antimicrobials may
be classified as “traditional” or “naturally occurring” (Davidson, 2001). A
number of traditional antimicrobials,
e.g., acetic acid and benzoic acid, are
approved for use in foods by most international regulatory agencies. Antimicrobials approved for use in the
NOVEMBER 2002 • VOL. 56, NO. 11
Sanitizers, as defined by the Environmental Protection Agency, are “pesticide
products that are intended to disinfect
or sanitize, reducing or mitigating
growth or development of microbiological organisms including bacteria, fungi
or viruses on inanimate surfaces in the
household, institutional, and/or commercial environment” (40 Code of Federal Regulations [CFR] 455.10). Sanitizers used by food manufacturers include chlorine and chlorine derivatives,
iodine derivatives, quaternary ammonium compounds, acid-anionic sanitizers,
hydrogen peroxide, peroxyacetic acid,
and acidified sodium chlorite (21 CFR
178.1010). Sanitizers are generally used
to inactivate target microorganisms on
the food contact surfaces of cleaned
food processing and food service equipment. A relatively novel use of these
compounds is for the inactivation of
microorganisms on raw, unprocessed
food products, e.g., meat and poultry
carcasses and fresh fruits and vegetables.
Responses of Microorganisms to
Antimicrobials and Sanitizers
If a population of microorganisms is
exposed to a sufficiently high concentration of an antimicrobial compound,
susceptible cells will be killed. However,
some cells may possess a degree of natural resistance or they may acquire it later
through mutation or genetic exchange
and will, therefore, survive and grow
(Bower and Daeschel, 1999). To fully
understand antimicrobial resistance,
one must understand the mechanisms
of action and/or the specific targets of
an antimicrobial within a microorganism. For example, antibiotics used for
therapeutic purposes often have specific
target sites in a microbial cell and the
development of resistance to these compounds is the result of changes in these
target sites. These changes may include
inactivation or modification of the antibiotic by enzymes within the cell, absence of or bypassing of an enzymatic or
metabolic step targeted by the antibiotic, impaired uptake or efflux of the antibiotic, modification of the antibiotic
target site, or overproduction of a target
molecule (Russell et al., 1997). Unfortunately, while we know a great deal about
the mechanisms of action and resistance
to antibiotics used therapeutically, the
precise mechanisms and targets of most
food antimicrobials and sanitizers remain a mystery. Therefore, we are less
able to predict and/or understand poVOL. 56, NO. 11 • NOVEMBER 2002
toplasmic membrane of microorganisms
tential resistance to these groups of
more effectively in the protonated form,
compounds.
they are most effective in their undissociThe resistance responses of microorated form (Davidson, 2001). Therefore,
ganisms to antimicrobials or sanitizers
the pKa (the pH at which 50% of the acid
may be innate, apparent, or acquired.
Innate resistance is a chromosomally
is in the undissociated form) of these
controlled property that is naturally ascompounds is important in selecting a
sociated with a microorganism. Differparticular compound for a specific appliences in resistance to antimicrobials occation. The lower the pH of a food prodcurring among different types, genera,
uct, the greater the proportion of acid in
species, and strains of microorganisms
an undissociated form, and the greater
under identical environmental condithe antimicrobial activity. Polarity is antions and antimicrobial concentrations
other important factor that affects apparare most likely controlled innately.
ent activity (Davidson, 2001). Polarity reMechanisms of innate resistance may
lates both to the ionization of the moleinclude cellular barriers preventing encule and the contribution of any hydrotry of the antimicrobial (e.g., the outer
carbon side groups or hydrophobic parmembrane of Gram-negative bacteria
ent molecules. Antimicrobials must be liand teichoic acids contained within
pophilic and soluble in the aqueous
Gram-positive bacteria), cellular efflux
phase to attach and pass through the cell
(i.e., mechanisms that pump commembrane. Microorganisms exposed to
pounds out of the cell), lack of a biofood antimicrobials in lipid-containing
chemical target for antimicrobial attachfood systems will demonstrate apparent
ment or microbial inactivation, and inincreased resistance due to the solubilizaactivation of antimicrobials by microbition or binding of the antimicrobials by
al enzymes (Bower and Daeschel, 1999).
the lipids.
Apparent resiscontinued on page 72 c
tance is related to
assay or application Table 2—Sources of some naturally occurring
conditions. As with
compounds with potential for food use or approved for
any preservation
use as food antimicrobials
technique, susceptibility to antimicroSource
Antimicrobial
bials is dependent
upon the condiAnimals/insects
tions of the applica- Milk
Lactoperoxidase system
tion. The presence
Milk, eggs
Lysozyme
of interacting stress
Milk
Lactoferrin
conditions (e.g.,
low pH, high temHoney
Glucose oxidase
peratures, high
Plants
pressure) may inSpices, herbs
Essential oils, phenolics, isoprenoids
crease or decrease
the measured resisOnions, garlic, horseradish
Sulfur compounds
tance of a microorBrassica (mustard,
Isothiocyanates
ganism. Food comBrussels sprouts, etc.)
position often has a
Grapefruit seed
Grapefruit seed extract
major influence on
Hops
Hop oils
the apparent activity of food antimiMicroorganisms
crobials, especially
Nisin, lacticin
organic acids. Food Lactococcus
Pediococcus
Pediocin
pH is the most universally important
Lactobacillus
Lactocin, helveticin, sakacin, bavaricin,
curvacin
factor that influences the effectiveness
Leuconostoc
Leucocin, mesentericin
of food antimicroCarnobacterium
Carnocin
bials. Many food
Streptomyces
natalensis
Natamycin
antimicrobials are
Microgard® (Rhodia Inc., Cranbury, N.J.),
weak acids. Because Fermentation products
Alta® (Quest International, Hoffman
these acids are able
Estates, Ill.)
to penetrate the cy-
FOODTECHNOLOGY 71
Scientific Status Summary
Acquired resistance results from genetic changes in the microbial cell
through mutation or acquisition of genetic material from plasmids (Russell,
1991). Because antibiotics used for therapeutic purposes generally have specific
target sites in microbial cells, they have
greater potential to result in mutations
and development of acquired resistance.
In contrast, antimicrobials used nontherapeutically and sanitizers are generally non-specific, and the development
of resistance to these compounds is
caused primarily by innate factors (Russell et al., 1997).
Bacterial Stress Responses
Food preservation processes are designed to either inhibit the growth of or
inactivate bacteria, depending upon the
type and severity of the process used.
Thus, food preservation exposes bacteria to both lethal and sub-lethal stresses.
Bacteria may have different mechanisms
for surviving these external environmental stresses. For example, the formation of endospores in response to stress
is a survival strategy for Bacillus and
Clostridium species. Bacteria that cannot
form endospores undergo other significant physiological changes that enhance
their ability to survive environmental
stressors. Regardless of the specific microbial strategy, genetic regulatory modification is involved. Common genetic
regulatory factors, called Sigma (s) factors, are frequently involved in enhanced
stress resistance. Sigma factors produced
in response to a stress bind to core microbial RNA polymerase, conferring different promoter specificities and leading
to the production of stress proteins
which protect the cell from the stress.
RpoS, for example, is a regulatory factor
required for transcriptional activation
of a large number of genes required for
tolerance to environmental stresses, including growth phase-dependent acid
tolerance. Rees et al. (1995) found that
rpoS-deficient mutants were highly sensitive to food processing conditions
compared with non-mutants. Abee and
Wouters (1999) prepared a very good
review of studies involving stress responses of foodborne bacterial pathogens.
Resistance of Microorganisms to
Traditional Antimicrobials
Benzoic acid and its salts were one of
the first groups of antimicrobials approved for application to foods in the
72
FOODTECHNOLOGY
United States. The primary application
of benzoic acid and benzoates is to inhibit yeasts and molds in acidic foods.
Differences in microbial resistance to
benzoates occur as a result of differences
in innate tolerance. Because benzoates
are used primarily as antifungal agents,
one might conclude that bacteria are
generally more resistant to the compounds than fungi. In fact, however,
bacteria are quite variable in their resistance to benzoates. Benzoates are used
primarily as antifungals because: (1)
they function best in the undissociated
state, which is the predominant form of
the compound at low pH in high acid
foods; and (2) fungi are the primary
spoilage microorganisms in acidic
foods. Therefore, the innate resistance of
yeasts and molds to benzoates is of
greater concern than that of bacteria. A
number of yeasts, including Schizosaccharomyces pombe and Zygosaccharomyces bailii, have been observed to grow in
the presence of about 500 µg/mL benzoic acid (Warth, 1985). Other yeasts, including Pichia membranefaciens and
Byssochlamys nivea, are also known to
be resistant to benzoates (Chipley,
1993).
Warth (1988) suggested that the
mechanism by which yeasts develop resistance to weak acidic antimicrobials,
including propionic as well as benzoic
acids, is related to membrane permeability and the ability of the cells to continuously pump antimicrobials out of
the cell. Some microorganisms on the
other hand have innate resistance to benzoates because they metabolize the compounds. These bacteria—Bacillus,
Pseudomonas, Corynebacterium, Micrococcus, and the mold Aspergillus—degrade benzoic acid through their b-ketoadipate pathway, in which benzoic
acid is converted to succinic acid and
acetyl Coenzyme A (Chipley, 1993).
Few studies examine the potential for
acquired resistance to benzoic acid. Warth (1988) incubated a variety of yeasts,
including Candida krusei, Hansenula
anomala, Kluyveromyces fragilis, Kloeckera apiculata, Saccharomyces cerevisiae,
Saccharomycodes ludwigii, S. pombe and
Z. bailii, in the presence of either 0.25
mM (31 µg/mL) or 2 mM (244 µg/mL)
benzoic acid. The minimum inhibitory
concentration (MIC) or lowest concentration preventing growth for unexposed cells was significantly lower for
cells exposed to these concentrations of
benzoic acid than for cells previously ex-
posed to sub-inhibitory concentrations
of benzoic acid. Pre-exposure to benzoic
acid caused a 1.4 to 2.2 fold increase in
MIC, with Z. bailii and S. pombe exhibiting the greatest MIC increases. The
proposed resistance mechanism was increased cellular efflux. There was no evidence to indicate any increased resistance due to mutation nor any evidence
that the resistance was stable. Further,
there is little or no evidence in the literature of acquired bacterial resistance to
benzoic acid.
Sorbic acid has been used as an antimicrobial in foods in the United States
since the 1940s when it was patented for
use in foods and on packaging to retard
spoilage by molds (Sofos and Busta,
1993). Innate resistance to sorbate is
demonstrated by bacteria, including catalase-negative lactic acid bacteria, Sporolactobacillus, some Pseudomonas, yeasts
(including Brettanomyces, Candida, Saccharomyces, Torulopsis, and Z. bailii),
and molds (including Aspergillus, Fusarium, Geotrichum, Mucor, and Penicillium) (Sofos and Busta, 1993; Warth,
1985). As with benzoic acid, some microorganisms can metabolize sorbic
acid. Molds isolated from cheese, including seven Penicillium species, exhibited growth in the presence of and degradation of 0.3 to 1.2% sorbate (Finol et
al., 1982). Penicillium puberulum and
Penicillium cyclopium were the most resistant species evaluated. Marth et al.
(1966) demonstrated that Penicillium
species isolated from cheese produced
1,3 pentadiene, which has a kerosene
off-odor, from sorbic acid. Sorbic acid is
also degraded by Mucor species to 4hexenol and by Geotrichum species to 4hexenoic acid and ethyl sorbate (Liewen
and Marth, 1985). High numbers of lactic acid bacteria can produce ethyl sorbate, 2,4-hexadien-1-ol, 1-ethoxyhexa2,4 diene, 5-hexadien-1-ol, and 2-ethoxyhexa-3,5 diene in sorbic acid-treated
red wine (Liewen and Marth, 1985). The
2,4 hexadien-1-ol metabolic product
can cause “geranium” type off-odors in
wines and fermented vegetables (Liewen
and Marth, 1985; Sofos and Busta,
1993).
As for benzoic acid, there is little evidence of acquired resistance to sorbic
acid. Warth (1977) observed that Z.
bailii grown in the presence of sorbic
acid acquired resistance to subsequent
exposure to the compound. Schroeder
and Bullerman (1985) found little or no
increase in the resistance of Penicillium
NOVEMBER 2002 • VOL. 56, NO. 11
digitatum or Penicillium italicum when
exposed to increasing concentrations of
sorbic acid. Bills et al. (1982) also investigated acquired resistance to sorbic acid
using the osmotolerant yeast, Saccharomyces rouxii. The yeast was pre-conditioned by growth in the presence of
0.1% sorbic acid for four transfers. Preexposure significantly increased resistance of cells subsequently exposed to
0.1% sorbic acid, as evidenced by shorter lag times and/or shorter time to stationary phase.
To combat the effects of sorbic and
other organic acids, yeasts have several
mechanisms by which they can develop
resistance. One mechanism for acquired
resistance that has been demonstrated
among yeasts is the triggering of an inducible, energy-requiring system that
increases sorbic acid efflux (Bills et al.,
1982; Warth, 1977). However, resistance
of yeasts to sorbic acid and other weak
acids probably involves more than one
system (Brul and Coote, 1999). The
mechanism by which organic acids inhibit microorganisms involves passage
of the undissociated form of the acid
across the cell membrane lipid bilayer.
Once inside the cell, the acid dissociates
because the cell interior has a higher pH
than the exterior. Protons generated
from intracellular dissociation of the organic acid then acidify the cytoplasm
and must be extruded to the exterior.
Yeasts use the enzyme, H+-ATPase, along
with energy in the form of ATP to remove excess protons from the cell. Inhibition and/or inactivation may be due to
eventual loss of cellular energy or inactivation of critical cellular functions due
to low intracellular pH.
Another mechanism used to prevent
depletion of energy pools involves the
induction of a membrane protein that
can decrease the activity of the ATPase
to conserve energy (Brul and Coote,
1999). In addition, exposure of S. cerevisiae to sorbic acid can strongly induce a
membrane protein ATP-binding cassette
transporter (Pdr12), which is a “multidrug resistance pump” that confers resistance by mediating energy-dependent
extrusion of anions (Piper et al., 1998).
Mutants without the transporter are hypersensitive to sorbic, benzoic, and propionic acids. One problem with extruding anions and protons is the potential
for recombination in the extracellular
medium, thus allowing them to reenter
the cell. To prevent the futile cycle allowing the acid back into the cell, adaptVOL. 56, NO. 11 • NOVEMBER 2002
ed yeasts apparently reduce diffusion
and passage of the weak acids into the
cell, most likely by altering cell membrane structures (Brul and Coote, 1999).
Similar mechanisms likely also exist for
bacteria that are capable of developing
resistance to sorbic or other organic acids. Considering the length of time that
sorbic and benzoic acids have been applied to food products it would seem,
however, that the development of acquired resistance by spoilage and pathogenic microorganisms is very rare or
non-existent.
Pre-exposure to sub-inhibitory concentrations of other food antimicrobials
has demonstrated varying resistance responses by microorganisms. For example, Moir and Eyles (1992) compared
the effectiveness of methyl paraben and
potassium sorbate on the growth of four
psychrotrophic foodborne bacteria—
In contrast to antibiotics
used for therapeutic
purposes, microbiologically derived antimicrobials
generally have a much
narrower spectrum
of activity. . . .
Aeromonas hydrophila, L. monocytogenes,
Pseudomonas putida and Yersinia enterocolitica. They observed little or no adaptation when cells were exposed to subinhibitory concentrations of antimicrobials. Bargiota et al. (1987) examined
the relationship between lipid composition of S. aureus and resistance to parabens. Differences in total lipid, phospholipids, and fatty acids were found for
S. aureus strains that were relatively resistant and a strain that was sensitive to
parabens. The paraben-resistant strain
had a higher percentage of total lipid,
higher relative percentage of phosphatidyl glycerol, and decreased cyclopropane
fatty acids compared with the sensitive
strains. Bargiota et al. suggested that
these changes could influence membrane fluidity and, therefore, adsorption
of the parabens to the membrane. Juneja and Davidson (1993) altered the lipid
composition of L. monocytogenes by
growth in the presence of added fatty
acids (C14:0, C18:0, and C18:1). Growth
of L. monocytogenes in the presence of
exogenously added C14:0 or C18:0 fatty
acids increased resistance of the cells to
parabens. Growth in the presence of
C18:1, however, increased sensitivity to
the antimicrobial agents. Thus, a correlation exists between lipid composition
of the L. monocytogenes cell membrane
and susceptibility to antimicrobial compounds.
Resistance of Microorganisms to
Naturally Occurring Antimicrobials
Most of the attention on acquired resistance to naturally occurring antimicrobials has been focused on microbiologically derived antimicrobials. The
probable reason for this is the similarity
in form and/or ability to kill target cells
that some of these compounds have to
medically important antibiotics. Because of the similarities, it has been suggested that use of microbiologically derived antimicrobials in foods may result
in the development of acquired resistance to the compounds themselves or
possibly cross resistance to antibiotics
used in human medicine. In contrast to
antibiotics used for therapeutic purposes, microbiologically derived antimicrobials generally have a much narrower
spectrum of activity, i.e., affecting limited types of target microorganisms, and
often having different mechanisms
which may reduce chances for acquired
resistance.
Two microbiologically derived antimicrobials that have been studied for
their impact on the development of acquired resistance are natamycin and nisin. Natamycin, formerly called pimaricin, is an antifungal produced by Streptomyces natalensis that is effective
against nearly all molds and yeasts but
which has little or no effect on bacteria.
Natamycin has no medical uses; however, it is used primarily as an antifungal
agent on cheese. De Boer and StolkHorsthuis (1977) investigated the potential for development of resistance to
natamycin among fungi. They reported
no evidence of resistant fungi in cheese
warehouses where natamycin was used
for periods of up to several years. They
also attempted to induce tolerance in 26
strains of fungi by transferring each culture 25-31 times in media containing
concentrations of natamycin equal to
and greater than the MIC. Following
multiple transfers, the MIC increased in
only 8 of 26 strains by a maximum of 4
FOODTECHNOLOGY 73
Scientific Status Summary
µg/mL. De Boer and Stolk-Horsthuis
concluded that lack of increased resistance among fungi was due to the lethal
(as opposed to static) activity of the
compound along with the compound’s
instability over time.
Nisin is a polypeptide composed of
34 amino acids that is produced by certain strains of Lactococcus lactis ssp. lactis. Nisin has a narrow spectrum of activity affecting primarily vegetative cells
and spores of Gram-positive bacteria.
Susceptible strains are found among lactic acid bacteria, Bacillus, Clostridium,
Listeria, and Streptococcus. The peptide
alone generally does not inhibit Gramnegative bacteria, yeasts, or molds. The
mechanism of antimicrobial action of
nisin against vegetative cells includes
binding to the anionic phospholipids of
the cell membrane and insertion into
the membrane, resulting in pore formation. Disruption of the cytoplasmic
membrane causes efflux of intracellular
components and eventual depletion of
the proton motive force (PMF; Crandall
and Montville, 1998). Microorganisms
exhibiting resistance to nisin may inactivate the peptide via enzymatic action or
they may alter their membrane susceptibility (Montville et al., 2001). Streptococcus thermophilus, Lactobacillus plantarum, and certain Bacillus species that
produce the enzyme nisinase neutralize
the antimicrobial activity of the
polypeptide (Hoover and Hurst, 1993).
In addition, spontaneous nisin resistant
mutants, including L. monocytogenes, C.
botulinum, Bacillus species, and S. aureus, could occur via exposure of wildtype strains to nisin or transfer of
strains in media containing increasing
concentrations of nisin (Harris et al.,
1991; Mazzotta et al., 1997; Ming and
Daeschel, 1993; Montville et al., 2001).
L. monocytogenes resistant mutants,
which are stable, may occur at a rate of 1
in 106 to 108 (Harris et al., 1991; Ming
and Daeschel, 1993) or even lower
(Schillinger et al., 1998). Crandall and
Montville (1998) observed that nisin resistant strains of L. monocytogenes (NisR)
had altered phospholipid composition,
including decreased anionic phospholipid (cardiolipin and phosphatidylglycerol) and increased phosphatidylethanolamine in the cell membrane resulting in a decreased net negative charge
that could hinder binding of cationic
compounds such as nisin. In addition,
the cell membranes of NisR strains exhibited increased long chain fatty acids
74
FOODTECHNOLOGY
and reduced ratios of C15/C17 fatty acids, suggesting reduced fluidity and stabilization caused by reduced effect on
PMF (Ming and Daeschel, 1993; Mazzotta and Montville, 1997). These and
other changes suggest an alteration of
the cytoplasmic membrane to prevent
access by nisin.
The obvious implication of the emergence of pathogenic microorganisms resistant to bacteriocins is the potential
hazard in foods that are preserved exclusively by a single compound. To overcome this potential hazard, some researchers suggest using combinations of
bacteriocins or combinations of bacteriocins with other antimicrobials or preservation methods (Mulet-Powell et al.,
1998; Schillinger et al., 1998). In theory,
combinations of bacteriocins could be
successfully applied if the mechanisms
of action of the bacteriocins were different. However, even this strategy must be
validated for each combination. Crandall and Montville (1998) demonstrated
that L. monocytogenes ATCC 700302 was
both nisin- and pediocin-resistant.
Cross resistance among bacteriocins,
however, is variable. Rasch and Knøchel
(1998) found no cross resistance between nisin- and pediocin-resistant
strains of L. monocytogenes but they did
observe pediocin and bavaricin cross resistance.
Because microorganisms in foods are
often exposed to some variation of acidic environmental conditions, it is interesting to speculate whether acid adaptation of a microorganism could alter its
sensitivity to bacteriocins. Van Schaik et
al. (1999) investigated acid adaptation at
pH 5.5 and bacteriocin sensitivity and
found that acid adapted L. monocytogenes was more resistant to nisin and lacticin 3147. The difference in resistance between the acid adapted and non-adapted cells was more noticeable with nisin
than with lacticin 3147. The potential
for this change in resistance in a food
system remains uncertain, however, because the experiment was done in a microbiological medium (tryptic soy broth
supplemented with 0.6% yeast extract).
The most important question concerning the potential for microorganisms to acquire resistance to bacteriocins is whether such resistance conveys a
natural advantage over non-resistant
strains in food systems. Mazzotta et al.
(2000) demonstrated that nisin-resistant
strains of L. monocytogenes and C. botulinum were not as resistant as wild-type
strains to other traditional food antimicrobials including sodium chloride, sodium nitrite and potassium sorbate.
Dykes and Hastings (1998) observed
that leucocin- and sakacin-resistant L.
monocytogenes B73 had a reduced
growth rate in a microbiological growth
medium (brain heart infusion broth)
without bacteriocin than bacteriocinsensitive strains. In addition, NisR strains
failed to compete with bacteriocin-sensitive strains when grown in mixed populations, even at a 1:1 ratio. Dykes and
Hastings concluded that the bacteriocin-resistant phenotype of L. monocytogenes B73 was not likely to become stable in natural populations. Gravesen et
al. (2002) also observed that pediocinresistant L. monocytogenes frequently exhibited a reduced growth rate and extended lag phase in a microbiological
broth medium compared to wild-type
cells. However, nisin-resistant L. monocytogenes strains had fewer and less pronounced growth rate reductions. Interestingly, pediocin- and nisin-resistant
strains were no more stress susceptible
(pH, salt, low temperature) than sensitive strains, and they grew equally well
in a model sausage system as the parent
strains. Mazzotta and Montville (1999)
demonstrated that nisin-resistant C.
botulinum 169B spores have similar heat
resistance patterns as wild type spores.
Therefore, while acquired resistance to a
single bacteriocin does not appear to
automatically confer resistance to other
antimicrobials or preservative treatments nor any natural advantage for a
population in the absence of the inhibitor, more research in food systems is
definitely warranted. For a more extensive review on naturally occurring antimicrobials, readers are referred to Sofos
et al. (1998).
Resistance of Microorganisms
to Sanitizers
Certain microorganisms, e.g., bacterial spores and Cryptosporidium, have
innate chlorine resistance. Microorganisms may also develop acquired resistance following exposure to chlorine.
Conditions that may lead to the development of resistance include application of sub-lethal concentrations of
chlorine used in error or, more likely,
neutralization of the compound during
use. For example, the antimicrobial activity of hypochlorites is significantly reduced by organic matter and high pH
(Cords and Dychdala, 1993). Mokgatla
NOVEMBER 2002 • VOL. 56, NO. 11
et al. (1998) studied the chlorine resistance of Salmonella isolates obtained
during various stages of poultry processing. They observed that some were
resistant to hypochlorous acid (defined
as growth in the presence of 72 µg/mL
hypochlorous acid). The frequency of
isolates exhibiting chlorine resistance
differed depending upon the processing
line location, but resistant strains occurred at most sites, including scalding,
plucking, and final packaged product.
The mechanisms of chlorine resistance
identified for Salmonella include catalase production, decreased activity of
membrane-bound dehydrogenases, and
decreased DNA damage (Mokgatla et al.,
2002). These alterations would result in
reduced hydroxy radicals and singlet oxygen, both of which react with hypochlorous acid (the active form of hypochlorite sanitizers) to cause cellular
inactivation and improve DNA repair.
Mokgatla et al. (1998) initially recommended that in poultry processing, concentrations of chlorine sufficient to ensure that Salmonella are eradicated and
not subjected to sub-lethal concentrations must be used. It is now common
practice to also use sanitizers, such as
chlorine, in the wash water for fresh and
minimally processed fruits and vegetables (Beuchat, 1996; Cherry, 1999).
Chlorine and other sanitizers, however,
do not necessarily eliminate pathogens
from these products (Zhuang et al.,
1995), which could potentially lead to
the development of resistant strains.
Development of resistance to other
compounds commonly used as sanitizers in food processing environments
may also be possible. Pickett and Murano (1996) exposed L. monocytogenes to
sub-lethal concentrations of an acidic
anionic sanitizer, a chlorine-based sanitizer, an iodophor, a quaternary ammonium agent, lactic acid, citric acid, and
propionic acid before challenging the
cells to minimum inhibitory concentrations of each. Except for the acid anionic
(phosphoric acid/dodecylbenzene sulfonic acid) sanitizer, there was no difference in susceptibility among the cells. L.
monocytogenes developed resistance to
the MIC of the acidic anionic (500 µg/
mL) when challenged after an initial
shock of 350 µg/mL with the same sanitizer. While citric acid did not produce
resistant cells at the test pH of 2.8, when
the pre-exposure pH was raised to 5.0,
the acid yielded cells that survived exposure to the MIC (0.4%). Pickett and
VOL. 56, NO. 11 • NOVEMBER 2002
Murano suggested that pre-exposure of
the cells to the dissociated form of the
acid, but not the undissociated form,
caused L. monocytogenes to become resistant. Strains of L. monocytogenes isolated from poultry were shown to be resistant (MIC = 16 µg/mL) to the quaternary ammonium sanitizer, benzalkonium chloride (Lemaître et al., 1998). Resistance was plasmid-borne and could
be transferred to other Listeria and S.
aureus. The plasmid genes may code for
an energy-dependent efflux system.
These authors suggested that use of a
single type of sanitizer may allow for selection and persistence of resistant
strains.
When microorganisms are attached
to a surface or are part of a biofilm, they
are more resistant to antimicrobials
than are freely suspended (planktonic)
cells (Bower and Daeschel, 1999; Frank
There is no evidence that
proper use of sanitizers in
food manufacturing will
lead to development of
resistant microorganisms.
and Koffi, 1990; Oh and Marshall,
1996). The increased resistance may be
caused by a lack of adequate contact of
the antimicrobial with the microorganisms (Frank and Koffi, 1990) or to adsorption of the antimicrobial to the biofilm glycocalyx (a complex extracellular
polysaccharide material) (Cross, 1990).
Although data are lacking, it is also possible that resistance to sanitizers could
be acquired by microorganisms in biofilms through exposure to sublethal levels of the compounds.
Some studies suggest a relationship
between microbial resistance to sanitizers and microbial resistance to antibiotics used therapeutically. For example,
Russell and Day (1996) noted that cross
resistance exists between biocides and
antibiotics, and Russell (1997) reported
that antibiotic-resistant S. aureus and
Staphylococcus epidermidis developed
plasmid-mediated resistance to chlorhexidine and quaternary ammonium
compounds. Russell (1997) reported
that methicillin-resistant S. aureus
(MRSA) were significantly more resis-
tant to both povidine-iodine and hypochlorite than methicillin-sensitive
strains. Moken et al. (1997) suggested
that microorganisms may become resistant to antibiotics through exposure to
pine oil disinfectants. Moken et al. exposed Escherichia coli to pine oil disinfectant in a microbiological disk assay
and isolated resistant mutants. Pine oilresistant mutants were also more resistant to the medically important antibiotics tetracycline, ampicillin and
chloramphenicol than the parent strain.
These mutants overexpressed a gene that
triggered an increase in the general antimicrobial efflux pump. The mutants did
not have cross resistance to hydrogen
peroxide, hypochlorite, or a quaternary
ammonium compound. The authors
suggested that constant use of disinfectants in the home may increase the development of resistance to antibiotics
used therapeutically.
There is no evidence that proper use
of sanitizers in food manufacturing will
lead to development of resistant microorganisms. However, with increasing reliance on and use of sanitizers on food
handling equipment, in food processing
environments, and on raw products, the
potential for emergence of such resistant
microorganisms does exist. Therefore,
the potential for development of resistant
strains must continue to be evaluated.
Resistance of Microorganisms to
Other Processing Conditions
It has been shown repeatedly in laboratory situations that bacteria can become resistant to certain environmental
factors under conditions that would
normally be considered lethal to the organism (Bower and Daeschel, 1999). For
example, E. coli O157:H7, Salmonella
Typhimurium, and L. monocytogenes
can become more acid resistant and
possibly more resistant to other stresses
(e.g., heat, osmotic pressure), if subjected to relatively mild acidity before exposure to more acidic conditions (Brudzinski and Harrison, 1998; Buchanan and
Edelson, 1999a; Garren et al., 1998; Leyer and Johnson, 1992, 1993; Leyer et al.,
1995; Mazzotta, 2001; O’Driscoll et al.,
1996; Ravishankar and Harrison 1999;
Wilde et al., 2000). Developed resistance
is referred to as tolerance, adaptation, or
habituation depending upon how the
microorganism is exposed to the stress
and the physiological conditions that
lead to enhanced survival (Buchanan
and Edelson, 1999b). In addition, pro-
FOODTECHNOLOGY 75
Scientific Status Summary
duction of acidic conditions by the microorganism itself can produce acid tolerance. For example, growth of E. coli in
an acidogenic broth (acid generating)
(e.g., tryptic soy broth [TSB] + glucose)
produced cells that expressed an acid resistance response while cells grown in a
nonacidogenic broth (TSB without glucose) did not (Buchanan and Edelson,
1999b).
Whether this occurs in actual food
environments during processing situations or in the product itself is intriguing, but largely unanswered. If pH resistance is the issue, then pH levels typically found in foods of concern should be
evaluated. Ravishankar and Harrison
(1999) found that L. monocytogenes exhibited an acid tolerance response when
it was acid adapted to pH 5.5 with lactic
acid and then challenged in acidified
skim milk at pH 3.5 and 4.0. When the
challenge pH of 4.5 was used, however,
there was no adaptive acid tolerance response. Because the pH of 4.5 is more
closely related to pH levels that might
occur in fermented products made from
skim milk, results based on this pH may
be the most meaningful. In apple, orange, and white grape juices, Mazzotta
(2001) found that the thermal resistance
of E. coli O157:H7, Salmonella, and L.
monocytogenes increased after acid adaptation. However, Mazzotta also pointed out that the typical pasteurization
process applied to these types of fruit
juices provides sufficient thermal inactivation of these pathogens, regardless of
whether or not they have enhanced thermal resistance due to acid adaptation.
Many studies of acid shock and acid
adaption of bacteria have been conducted. Most studies have evaluated these responses over a relatively short period of
time (typically a few hours). Within
foods and on food contact surfaces,
adaptive alteration of cells might be of
more concern if the resistance is sustained by the adapted cells. The greater
the degree of severity of the antimicrobial challenge, the less likely it will be for
the microorganism to survive for extended periods, even if it is adapted.
Numerous product and antimicrobial
combinations are possible in foods. Under certain scenarios, there may be reason
for concern when considering whether or
not bacteria can acquire some degree of
resistance to a particular antimicrobial.
Direct acidification of a food or food ingredient may shock microflora so they
become more acid resistant. Fermenta76
FOODTECHNOLOGY
tion of foods, however, may lead to
somewhat different situations. Lactic acid
bacteria can lower the pH of a substrate
gradually over time, likely resulting in a
pH gradient rather than a sharp change
in pH as would be expected with direct
acidification. Leyer et al. (1995) reported
an acid adaptive response in E. coli
O157:H7 which enhanced its survival in
fermented sausage (pH 5.6).
The use of acidic antimicrobial
sprays on the surfaces of meat carcasses
has become very common (Dickson,
1995). One could ask if bacteria on the
meat surface become more acid resistant
when they are exposed to a low concentration of a weak acid (e.g., <3%) solution. Whether or not the exposure of
microorganisms to acids on substrates
such as meat is sufficient to result in acquired resistance is largely unknown at
this time. One study by Van Netten et al.
(1998), however, demonstrated a lack of
increased resistance to lactic acid for E.
coli O157:H7, S. Typhimurium, S. aureus, and C. jejuni when acid adapted
cells were inoculated on pork bellies and
treated with 2% lactic acid as a sanitizer.
However, most other studies related to
acquired stress response have been done
either in laboratory culture media with
lower than typically encountered concentrations of antimicrobials, or in foods
under conditions which might be only
marginally similar to actual situations.
In a study using laboratory culture
broth, Duffy et al. (2000) reported that
E. coli O157:H7, which was adapted to
acid under mild acidic conditions, acquired cross protection to heat in an
acidic environment. They concluded
that this might have implications for fermented meat production and similar
processes, which are also given a thermal
process. However, since this study was
done in culture broth, it does not fully
mimic what could occur in the more
complex food matrix.
Several studies involving acid adapted microbes in heated and or dried meat
products have been conducted. In many
of these, acid adaptation did not offer
any enhanced protection to other environmental stresses to which the microorganism was subsequently exposed in
an actual food product. Calicioglu et al.
(2002) investigated the inactivation of
acid adapted and unadapted E. coli
O157:H7 during the processing of beef
jerky. They found that the survival rate
of E. coli O157:H7 varied depending on
the marinade formulation used in mak-
ing the product. Depending on the formulation, there was either no significant
effect on the survival rate between the
acid adapted and non-adapted cells or
the populations of acid adapted cells actually decreased at a more rapid rate
than the non-adapted populations. In
considering their results and those of
Calicioglu et al. (1997, 2001), and Riordan et al. (2000), Calicioglu et al. (2002)
offered the suggestion that heating
foods with a lowered pH and a low aw
may present a combination of preservation effects that overcomes any cross
protection benefit provided by acid adaptation.
In addition to looking at the possibility of L. monocytogenes becoming
more acid resistant through an acid tolerance response, Ravishankar and Harrison (1999) also conducted experiments to determine if acid adaptation of
the microorganism by cross protection
enhanced survival in the presence of an
activated lactoperoxidase system, a naturally occurring antimicrobial system in
milk. They observed that the survival
rates were similar for the acid adapted
and non-adapted cells at pH 4.5 both in
the presence and absence of an activated
lactoperoxidase system, indicating that
no cross protection was afforded the
acid adapted cells. In contrast, Leyer and
Johnson (1993) reported cross protection to an activated lactoperoxidase system with acid adapted S. Typhimurium
in a laboratory culture medium. The activity of the lactoperoxidase system can
vary depending on the medium, a possible explanation for the contrasting results. Another possible explanation for
the difference in the studies may be the
greater degree of acid tolerance exhibited by Salmonella than Listeria. The lactoperoxidase system may also be a less
effective antimicrobial toward Salmonella compared with Listeria.
Leyer and Johnson (1993) also reported that acid adapted S. Typhimurium offered cross protection against
heat, salt, and selected surface active
agents. O’Driscoll et al. (1996) found
that acid adapted L. monocytogenes also
exhibited increased resistance to heat,
cold, salt, selected surface active agents,
and ethanol. However, Leyer and
Johnson (1997) reported that when
populations of S. Typhimurium were
acid adapted at a pH of 5.0 to 5.8 the
sensitivity of the cells to hypochlorous
acid and iodine increased. They also investigated the mechanism of the inactiNOVEMBER 2002 • VOL. 56, NO. 11
vation by hypochlorous acid and concluded that, whether the cells were acid
adapted or not, the mechanism involved
changes in membrane permeability, inability to maintain or restore energy
charge, and probably oxidation of essential cellular components. They proposed
that acid pre-treatment in a food plant
sanitation program may thus enhance
the efficiency of halogen sanitizers.
Using a L. monocytogenes isolate
from a food processing plant drain,
Taormina and Beuchat (2001) investigated the survival and heat resistance of
the microorganism after exposure to alkaline pH (up to pH 12.0) and after exposure to chlorine (up to 6.0 mg of free
chlorine per liter). The alkaline stress
enhanced the resistance of L. monocytogenes to thermal processing conditions
of 56 and 59°C. In contrast, exposure of
the cells to chlorine resulted in populations that were more sensitive to heating
at 56°C. Based on these results, there are
differences in the cross protection offered after exposure to alkaline pH depending on whether an alkaline- or
chlorine-based sanitizer is used in sanitation routines.
Conclusions
Because evidence exists that microorganisms can acquire varying levels of
resistance or tolerance to environmental stresses, there is some concern that
this might provide protection for foodborne pathogens against antimicrobials
and preservation processes. Development of resistance to manufacturing
and processing treatments could occur
at numerous points in a food production system and could influence preservation treatment efficacy. However, before decisions can be made with confidence concerning the development of
tolerance or resistance among foodborne pathogens, it is critical to acquire
data that is relevant to real food processing situations. In addition, conditions
most representative of those in actual
products or food processing situations
must be included in experimental protocol. For example, as was noted by Leyer and Johnson (1993), the physiological
state of foodborne pathogens used in
challenge studies in food and in evaluating Hazard Analysis Critical Control
Point programs is an important consideration because the effectiveness of control measures may vary with varied microbial physiological states.
A major problem with use of antimiVOL. 56, NO. 11 • NOVEMBER 2002
crobial combinations, however, is the
general lack of knowledge about food antimicrobial mechanisms. Without this information, antimicrobials cannot be applied effectively to achieve synergistic interactions. More research is needed to determine: (1) the frequency and mechanisms of resistance to food antimicrobials and process stresses in food systems,
(2) mechanisms of action of traditional
and naturally occurring food antimicrobials, and (3) application strategies to
minimize tolerance or resistance development. Simple methods for overcoming
the potential for development of acquired resistance include using appropriate antimicrobials, avoiding the use of
sub-lethal concentrations of antimicrobials, using combinations of antimicrobials
for environmental or process controls
(such as hurdle technology) and using
combinations of antimicrobials that have
different mechanisms. Although data
concerning development of resistance to
food antimicrobials is scarce, antimicrobial resistance does not appear to be a
phenomenon that would have a major
negative impact on public health.
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NOVEMBER 2002 • VOL. 56, NO. 11