Perspectives on the Use of Organic Acids

and Short Chain Fatty Acids as Antimicrobials

S. C. Ricke1
Department of Poultry Science, Texas A&M University, College Station, Texas 77843-2472
Although this situation has implications regarding the
use of organic acids, it may only apply to circumstances
in which reduced acid levels have induced resistance and
virulence mechanisms in exposed organisms. Evaluating
effectiveness of organic acids for specific applications requires more understanding general and specific stress
response capabilities of foodborne pathogens. Development and application of molecular tools to study pathogen behavior in preharvest and postharvest food production environments will enable dissection of specific bacterial genetic regulation involved in response to organic
acids. This could lead to the development of more targeted strategies to control foodborne pathogens with organic acids.

(Key words: organic acid, antimicrobial, short-chain fatty acid, stress)

2003 Poultry Science 82:632639

given the equally intensive public concern regarding

prevalence of foodborne pathogens in food animals during preharvest and postharvest phases of animal production, limiting pathogen dissemination with antimicrobials
remains a desired goal. This has lead to somewhat of
a renaissance in research activity on the discovery and
application of nonantibiotic chemical compounds capable
of either killing microorganisms outright or at the very
least retarding growth sufficiently to limit their dissemination.

INTRODUCTION
Antibiotic supplementation in farm animal diets to improve animal growth performance and efficiency of feed
conversion, historically, has been theorized as a means
of establishing food animals as reservoirs for antibioticresistant pathogens isolated from humans (Smith, 1968;
Levy et al., 1976; Armstrong, 1984; Neu, 1992). Given
the rising concerns associated with increased antibiotic
resistance in bacterial pathogens making treatment of
clinical diseases more difficult, antibiotic use in animal
management in the U.S. and Europe has become more of
a controversial issue (Glynn et al., 1998; Monnet, 1999;
Koutsolioutsou et al., 2001). This has resulted in a heightened awareness regarding exposure of foodborne pathogens to therapeutic levels of antibiotics during preharvest
phases of food animal production (Aarestrup and Wegener, 1999; Threlfall et al., 2000). Such concern necessitates
a certain perceived inevitability for potential decreases
in the uses of traditional antibiotics for routine nonclinical
applications in animal management practices. However,

ORGANIC ACIDS
Classification and Uses
Organic acids have a long history of being utilized
as food additives and preservatives for preventing food
deterioration and extending the shelf life of perishable
food ingredients. As a group these compounds primarily
include the saturated straight-chain monocarboxylic acids
and their respective derivatives (unsaturated, hydroxylic,
phenolic, and multicarboxylic versions) and are often generically referred to as fatty acids, volatile fatty acids, or

2003 Poultry Science Association, Inc.

Received for publication October 9, 2002.
Accepted for publication December 5, 2002.
1
To whom correspondence should be addressed: sricke@poultry.
tamu.edu.

Abbreviation Key: ATP = adenosine triphosphate; SCFA = shortchain fatty acids.

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ABSTRACT Organic acids have a long history of being

utilized as food additives and preservatives for preventing food deterioration and extending the shelf life of
perishable food ingredients. Specific organic acids have
also been used to control microbial contamination and
dissemination of foodborne pathogens in preharvest and
postharvest food production and processing. The antibacterial mechanism(s) for organic acids are not fully understood, and activity may vary depending on physiological
status of the organism and the physicochemical characteristics of the external environment. An emerging potential
problem is that organic acids have been observed to enhance survivability of acid sensitive pathogens exposed
to low pH by induction of an acid tolerance response and
that acid tolerance may be linked to increased virulence.

SYMPOSIUM: USE OF ANTIMICROBIALS IN PRODUCTION

Antibacterial Activities of Organic Acids

In general, potential bacterial targets of biocidal compounds include the cell wall, cytoplasmic membrane, and
specific metabolic functions in the cytoplasm associated
with replication, protein synthesis, and function (Denyer
and Stewart, 1998; Davidson, 2001). Although the antibacterial mechanism(s) for organic acids are not fully understood, they are capable of exhibiting bacteriostatic and
bactericidal properties depending on the physiological
status of the organism and the physicochemical characteristics of the external environment. Given the weak acid
nature of most of these compounds, pH is considered a
primary determinant of effectiveness because it affects the
concentration of undissociated acid formed (Davidson,
2001). It has been traditionally assumed that undissociated forms of organic acids can easily penetrate the lipid
membrane of the bacterial cell and once internalized into
the neutral pH of the cell cytoplasm dissociate into anions
and protons (Eklund, 1983, 1985; Salmond et al., 1984;
Cherrington et al., 1990, 1991; Davidson, 2001). Generation of both of these species potentially presents problems
for bacteria that must maintain a near neutral pH cytoplasm to sustain functional macromolecules. Export of
excess protons requires consumption of cellular adenosine triphosphate (ATP) and may result in depletion of
cellular energy (Davidson, 2001).
Other toxicity mechanisms have been proposed that
attribute membrane uncoupling capabilities for organic
acids. It has been speculated that organic acids interfere
with cytoplasmic membrane structure and membrane
proteins such that electron transport is uncoupled and
subsequent ATP production is reduced or that organic

acids serve as uncouplers that generally dissipate pH

and electrical gradients across cell membranes (Sheu and
Freese, 1972; Sheu et al., 1972, 1975; Freese et al., 1973;
Salmond et al., 1984; Russell, 1992; Axe and Bailey, 1995;
Davidson, 2001).
Russell (1992) has hypothesized that anion accumulation is the primary toxic effect of organic acids and that
some organisms are more resistant to organic acids because they are capable of allowing their internal pH to
decline. Less direct antibacterial activities have also been
attributed to organic acids and include interference with
nutrient transport, cytoplasmic membrane damage resulting in leakage, disruption of outer membrane permeability, and influencing macromolecular synthesis (Cherrington et al., 1991; Denyer and Stewart, 1998; Alakomi
et al., 2000; Davidson, 2001).
The mechanisms associated with these activities have
been difficult to establish due to the complex nature of
the interaction between energy dissipation and disruption
of ATP generation capabilities of the bacteria. Consequently, bactericidal concentrations of organic acids may
be due to the combination of dissipation of proton-motive
force and inability to maintain internal pH followed by
denaturation of acid-sensitive proteins and DNA. Sublethal concentrations may elicit their effects on overall cell
physiology and lead to responses such as enlargement of
bacterial cell size (Thompson and Hinton, 1996).

Factors that Influence Toxicity

Efficacy of Organic Acids
The significant affects and the toxicity of organic acids
(either directly or indirectly) for foodborne pathogens
have not been clearly elucidated. This is in part due to
complexities involved in designing and conducting the
experiments that precisely delineate all of the contributing factors. Direct assessment and comparison of antibacterial potency of individual acids can be elusive because
of the influence of the physical chemistry of respective
acids, the bacterial species in question, growth conditions
or media composition, and the growth phase (Cherrington et al., 1991; Lasko et al., 2000). In addition, specific
quantitative in vitro responses do not necessarily translate
to all in vivo possibilities for a particular organism. This
is partially due to the fact that many of the common
foodborne pathogens such as Salmonella spp. can grow
in a multitude of ecosystems and, thus, in vivo growth
conditions when exposure to organic acids occurs can
vary widely. It is entirely possible that during a life cycle,
Salmonella can grow aerobically and within a short time
frame be forced to switch to an anaerobic metabolism to
survive and potentially colonize a highly fermentative
environment such as the gastrointestinal tract. Under
these conditions not only would Salmonella be required to
survive high concentrations of SCFA produced by other
organisms, but it would also be generating and exporting
fermentation organic acids of its own. Distinguishing specific organic acid toxic mechanisms in foodborne pathogens will continue to be difficult to achieve until genetic

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weak or carboxylic acids Cherrington et al., 1991). Several

of these organic acid compounds are used as direct additives incorporated into human foods or can accumulate
over time as a consequence of the fermentation activity
of indigenous or starter cultures added to certain dairy,
vegetable, and meat products. In addition, acid sprays
have been incorporated as sanitizers during meat processing (Acuff et al., 1987; Cherrington et al., 1991; Dickson, 1992; Hardin et al., 1995; Dorsa, 1997). Some organic
acids, particularly the short-chain fatty acids (SCFA), acetate, propionate, and butyrate, are produced in millimolar
quantities in the gastrointestinal tracts of food animals
and humans and characteristically occur in high concentrations in regions where strictly anaerobic microflora are
predominant. In the food animal industry, organic acids
were originally added to animal feeds to serve as fungistats (Paster, 1979; Dixon and Hamilton, 1981), but in
the past 30 yr, formic and propionic acids and various
combinations have also been examined for potential bactericidal activity in feeds and feed ingredients contaminated with foodborne pathogens, particularly Salmonella
spp. (Khan and Katamay, 1969; Vanderwal, 1979; Williams, 1981; Hinton and Linton, 1988; Humphrey and
Lanning, 1988; Izat et al., 1990; McHan and Shotts, 1992;
Berchieri and Barrow, 1996; Thompson and Hinton, 1997).

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acids simply disappear in the gastrointestinal tract before

reaching locations inhabited by pathogens. Using radioactively labeled propionate, Hume et al. (1993b) demonstrated that most of the propionic acid originating from
the treated feed is metabolized and absorbed in the foregut of the chicken (crop, gizzard, and proventriculus) and
does not reach the small and intestine or the cecum in
sufficient quantities to be effective.

Bacterial Mechanisms of Resistance to

Antibacterial Activities of Organic Acids
In addition to the environmental factors that can influence the efficacy of organic acids as antimicrobial chemicals, inherent resistance of the target microorganism to
these compounds is also a factor (Davidson, 2001). Microbial sensitivity to antimicrobial agents determines relative
tolerance and is dependent on the type of organism (fungi,
virus, or bacteria), strain-to-strain variation and form (endospore versus vegetative cell) with vegetative cells being
more susceptible and bacterial spores being the most resistant (Heinzel, 1998). Metabolically, microbial resistance
may be transient in nature or expressed in a more stable
form, and mechanisms of resistance can include exclusion
of the antimicrobial chemical, secretion of the toxic compound, or metabolic detoxification (Heinzel, 1998).
Genetic encoding in bacteria for resistance to antimicrobial compounds can be fairly mobile within a population
of the same organism and can potentially be transferred
among populations of genetically different organisms.
Although less is known about organic acids, genetic
mechanisms in microorganisms for resistance to antibiotics and inorganic chemical-based disinfectants have been
well documented. Antibiotic resistance can be transmitted
among organisms by several mechanisms (Koutsolioutsou et al., 2001). Bacteria can become antibiotic resistant
by receiving and carrying antibiotic resistant encoding
genes on a plasmid that is replicated as an extrachromosomal genetic element during microbial multiplication
(Davies, 1994; Koutsolioutsou et al., 2001). In addition,
mutations can occur on the bacterial chromosome that
decrease susceptibility to antibiotic killing mechanisms,
or genes responsible for multiple resistances are turned
on by some sort of general stimuli (Nikaido, 1994; Spratt,
1994; Miller and Sulavik, 1996; Alekshun and Levy, 1997).
Although antibiotics have received the majority of the
focus, other compounds used to control microbial contamination in food production such as organic acids can
also elicit resistance responses in microorganisms (Chapman, 1998). Furthermore, the response of organisms to
these compounds may decide whether or not they survive
a variety of environmental stressors encountered in animal production and processing. Typical stresses encountered in food systems by foodborne pathogens include
deficiencies in biologically available iron, heat and cold
shock, oxidative stress, osmolarity stress, acid and alkaline pH, and nutrient starvation (Archer, 1996). The genetic regulation of many of these environmental stressors
is becoming better understood and in many cases may

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techniques can be applied to screen for differential physiological responses in selected mutants exposed to the
multitude of potential environmental backgrounds.
Efficacy of organic acids can also be highly influenced
by extrinsic factors associated with the environment that
they are being added to. An example of this complexity
is the use of propionic acid and its salt forms in animal
and poultry feeds as an anti-Salmonella and antifungal
compound. It has been suggested that the presence of
propionate in feeds limits Salmonella and fungal contamination in the ingredients added to feed and mixed feeds
during storage. However, several factors can alter the
effective concentration in the feed. Inconsistent antimicrobial effects of propionic acid on fungal populations in
feed has been attributed to possible buffering and conversion to its less active form by protein ingredients such as
soybean meal as well as batch differences in primary feed
components such as corn meal (Dixon and Hamilton,
1981; Tabib et al., 1984). Feed composition may also influence sensitivity of the organism. For example, there appears to be an interaction between water activity and
survival of salmonellae with more sustained survival occurring in populations adapted to lower water activities
(Mossel and Koopman, 1965; Liu et al., 1969; Carlson and
Snoeyenbos, 1970; Juven et al., 1984; Rouse et al., 1988).
This becomes a factor in evaluating organic acid efficacy
because it has been demonstrated that after 8 wk only
minimal reductions in salmonellae populations occur and
viable populations can be recovered from feed 3 to 18 mo
post inoculation (Williams and Benson, 1978; Davies and
Wray, 1996; Ha et al., 1998a,b). The resulting physiological
status of the pathogen contained in the feed over long
periods may also influence organic acid efficacy as bacterial cells in stationary phase can become more resistant
to various environmental stresses (Kolter et al., 1993).
Feeds treated with organic acids are also believed to
limit pathogen infestation once ingested by the animal.
Both formic and propionic acids have been reported in
several studies to reduce Salmonella, coliforms, and Escherichia coli in the small intestinal, cecal, and fecal contents
of chickens (Hinton and Linton, 1988; Rouse et al., 1988;
Izat et al., 1990; McHan and Shotts, 1992). It is presumed
that the presence of organic acids in feeds limits pathogen
colonization by conversion of these acids into their respective antibacterial forms once they have entered the digestive tract of the animal consuming the treated feed. However, Hume et al. (1993a) observed inconsistent reductions of Salmonella colonization in the crops and ceca of
chickens infected with a Salmonella marker strain. Several
factors can alter the effective antibacterial concentration of
an organic acid after the compound reaches the intestinal
tract. The pH of the gastrointestinal tract may be a factor
that influences the level of dissociated organic acid present since the average pH levels increase toward neutrality
as the small intestine and large intestine are traversed in
most nonruminant animal species (Wolin, 1974; Watkins
and Miller, 1983). Likewise, the rumen in most ruminant
species is generally buffered within a neutral pH range
(Hungate, 1966; Wolin, 1974). In addition, some organic

SYMPOSIUM: USE OF ANTIMICROBIALS IN PRODUCTION

involve highly intricate regulatory networks (Christman,

et al., 1985; Spector et al., 1986; Matin et al., 1989; Spector
and Cubitt, 1992; Kolter et al., 1993; Foster and Spector,
1995).
Adding to this complexity is the recent suggestion that
E. coli possesses extracellular sensing components that
may provide an early response to stress by the bacterial
cell and result in induction of the corresponding stress
systems inside the bacterial cell (Rowbury, 2001). There
is some indication that if stress induction occurs in a
bacterium when exposed to a single stressor, cross-protection can be manifested across multiple stresses and general tolerance of these environments is increased (Foster
and Spector, 1995a; Archer, 1996). Environmental changes
may also potentially be relayed among members of bacterial populations by cell-to-cell communication or quorum
sensing of diffusible low molecular weight signal compounds (Surette et al., 1999; de Kievit and Iglewski, 2000;
Withers et al., 2001).

Acid shock is a commonly occurring event experienced

by foodborne pathogens during exposure to food processing and preservation. It has been known for over 60
yr that bacteria possess metabolic capabilities to respond
to low external pH (Slonczewski, 1992). A more recent
observation is that acid-sensitive bacteria could adapt to
acid stress by induction of an acid tolerance response.
This response essentially involves growth of the acidsensitive microorganism in a moderately low pH environment that subsequently leads to survival when suddenly
exposed to what would normally be considered lethal
acidic conditions (Foster, 1995). This adaptation permits
the induction of genes involved in an acid-tolerance response and synthesis of a series of acid shock proteins
that are protective for extreme acidic conditions (Foster,
1999). Multiple acid-tolerant systems that are physiologically distinct have been identified in foodborne pathogens
such as Salmonella and E. coli and are classified as log
phase and stationary mechanisms (Foster, 1999). Foster
(2001) has further classified these in ascending order of
tolerance as log phase bacteria, acid-adapted log phase
bacteria, stationary phase bacteria, and acid-adapted stationary phase bacteria.
Weak organic acids such as the SCFA represent a potential acid stress exposure to foodborne pathogens due to
high concentrations and low pH. Uncharged protonated
weak acids are freely diffusable across membranes with
potential to lower internal pH (Bearson et al., 1997). It is
no surprise that an active area of research focus is the
relationship between SCFA and potential for induction
of acid-tolerance mechanisms. High concentrations (anywhere from 10 to 100 mM) of SCFA produced by anaerobic native intestinal microflora are frequently encountered by foodborne pathogens (Meynell, 1963; Hungate,
1966; Wolin, 1974; Cummings et al., 1987). Pathogens may
also encounter the SCFA in food products (100 to 300

mM) where they are widely used as preservatives due

to their antibacterial activities (Cherrington et al., 1991;
Hardin et al., 1995; Dorsa, 1997). The acid-tolerance system has been demonstrated to protect Salmonella against
lethal effects of organic acids and increase survival in
fermented foods (Leyer and Johnson, 1992; Baik et al.,
1996). Although it might be anticipated that SCFA at
acidic pH levels could induce acid tolerance, it has also
been shown that high concentrations of SCFA at neutral
pH are capable of inducing acid tolerance in E. coli and
S. typhimurium (Kwon and Ricke, 1998a,b; Arnold et al.,
2001). Kwon and Ricke (1998a,b) found that inorganic
acid resistance of S. typhimurium was increased after exposure to high concentrations of SCFA, and this SCFA-induced acid resistance was further enhanced by acidic pH,
anaerobiosis, and prolonged exposure to SCFA. Previous
exposure to SCFA also increased S. typhimurium survivors
against extreme acid (pH 3.0), high osmolarity (2.5 M
NaCl), and reactive oxygen (20 mM hydrogen peroxide)
and consequently could potentially cross-protect Salmonella from other environmental conditions that exist in
food animal and processed food products (Kwon et al.,
2000).
The interaction between acid shock and bacteria such
as Salmonella and E. coli, however, remains complicated
because growth and survival responses to SCFA appear
to be dependent on not only the growth phase of the
organism but atmospheric conditions (aerobic or anaerobic) used to recover survivors, salt form of the SCFA, and
perhaps initial concentration of the SCFA (Kwon et al.,
1998; Blankenhorn et al., 1999; Kwon and Ricke, 1999).
Future research will require examining growth and survival under more ecologically simulating conditions using techniques such as continuous culture to reproduce
the slow growth conditions and constant exposure to
fermentation typically occurring in the gastrointestinal
tract.
In addition, genetic tools that allow for precise dissection of all potential genes directly involved in SCFA and
stimulation of acid shock as well as peripheral systems
are required. Recent efforts with transposon mutagenesis
and gene array approaches have indicated that these
methods may be viable for identifying some of the prospective genes involved in SCFA-induced acid tolerance
(Arnold et al., 2001; Kwon et al., 2002).

Organic Acids and Pathogenesis

of Foodborne Bacteria
The concept of SCFA enhanced survivability to multiple environmental stresses has implications not only for
gastrointestinal ecology competitiveness of salmonellae
and foodborne pathogens in general, but, in the case of
SCFA, it raises practical issues regarding the use of organic acids as an intervention step in food processing.
Simply put this becomes a question of whether more
pathogenic subpopulations of a foodborne pathogen are
selected for after exposure to organic acids. Microbial
pathogenesis is a function of virulence expression in the

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Organic Acids and Induction

of Acid Tolerance

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enhance identification of signals for stimulation of virulence in the pathogen of interest and provide specific
targets for development of more optimal strategies to
control this foodborne pathogen.

Conclusions
An obvious prerequisite for successful efforts to minimize the impact of antibiotic resistance in the animal
industry is development and implementation of alternative antimicrobials. Organic acids have a long history of
use in the food industry as food preservatives and have
been demonstrated to be effective under a wide variety
of food processing conditions. Although there is indication that some tolerance by foodborne pathogens is possible, this tolerance may only be for particular circumstances in which exposure to the organic acid is less than
maximum and provides the organism an opportunity to
induce specific stress resistant and virulence mechanisms
that allow for resistance to more severe acidic conditions.
Improving effectiveness of organic acids will require
more understanding of general and specific stress response capabilities of foodborne pathogens. This would
include development and application of molecular tools
for studying pathogen behavior in microbial ecosystems
in a variety of preharvest and postharvest food production environments. Genetic techniques are now becoming
available to enable in depth dissection of the level of
interrelated regulation involved in the sensing and response of a pathogen to the respective environment. However, understanding the potentially high throughput of
resulting data may present the next challenge to devising
a practical strategy that anticipates potential problems
and implementing effective control measures.

ACKNOWLEDGMENTS
This review was supported by Hatch grant H8311 administered by the Texas Agricultural Experiment Station
and USDA-NRI grant number 2002-35201-11608.

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