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Applied and Environmental Microbiology, June 2001, p. 2410-2420, Vol. 67, No. 6
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2410-2420.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Influence of the Natural Microbial Flora on the Acid Tolerance
Response of Listeria monocytogenes in a Model System of
Fresh Meat Decontamination Fluids
John
Samelis,1
John N.
Sofos,1,*
Patricia
A.
Kendall,2 and
Gary
C.
Smith1
Center for Red Meat Safety, Department of
Animal Sciences,1 and Department of Food
Science and Nutrition,2 Colorado State
University, Fort Collins, Colorado 80523
Received 30 November 2000/Accepted 20 March 2001
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ABSTRACT |
Depending on its composition and metabolic activity, the natural
flora that may be established in a meat plant environment can affect
the survival, growth, and acid tolerance response (ATR) of bacterial
pathogens present in the same niche. To investigate this hypothesis,
changes in populations and ATR of inoculated (105 CFU/ml)
Listeria monocytogenes were evaluated at 35°C in water (10 or 85°C) or acidic (2% lactic or acetic acid) washings of beef
with or without prior filter sterilization. The model experiments were
performed at 35°C rather than lower (
15°C) temperatures to
maximize the response of inoculated L. monocytogenes in the washings with or without competitive flora. Acid solution washings were
free (<1.0 log CFU/ml) of natural flora before inoculation (day 0),
and no microbial growth occurred during storage (35°C, 8 days).
Inoculated L. monocytogenes died off (negative enrichment) in acid washings within 24 h. In nonacid (water) washings, the pathogen
increased (approximately 1.0 to 2.0 log CFU/ml), irrespective of
natural flora, which, when present, predominated (>8.0 log CFU/ml) by
day 1. The pH of inoculated water washings decreased or increased
depending on absence or presence of natural flora, respectively. These
microbial and pH changes modulated the ATR of L. monocytogenes at 35°C. In filter-sterilized water washings, inoculated L. monocytogenes increased its ATR by at least
1.0 log CFU/ml from days 1 to 8, while in unfiltered water washings the
pathogen was acid tolerant at day 1 (0.3 to 1.4 log CFU/ml reduction)
and became acid sensitive (3.0 to >5.0 log CFU/ml reduction) at day 8. These results suggest that the predominant gram-negative flora of an
aerobic fresh meat plant environment may sensitize bacterial pathogens
to acid.
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INTRODUCTION |
The initial microbial contamination
and the processing and storage conditions of meat affect the microbial
flora that dominates, which also affects survival and growth of
bacterial pathogens present (13, 27, 40, 47). The growth
and metabolic activity of these dominating organisms either leads to
spoilage (13, 42, 47) or contributes to preservation
(32, 40). Regardless of its spoilage or beneficial
potential, the population of the indigenous, nonpathogenic microbial
flora may be several logarithmic cycles higher than that of the
naturally occurring pathogens (40, 47). Thus, the natural
flora is usually expected to have a competitive advantage over
pathogens for nutrient uptake and may alter the intrinsic factors (pH,
water activity, nutrient catabolism, production of inhibitory
metabolites, etc.) of meat or other foods in ways that the survival and
growth of pathogens may be stimulated (19, 33, 34), not
affected (5, 34), or suppressed (4, 10, 11, 35, 40,
50). Similar associations may also develop in meat plant
environments where reservoirs of microbial contamination may be
established (41).
Although many predictive models have been developed with data derived
from experiments with pure pathogenic cultures in liquid media or
simulated food systems (12, 20, 36), recent studies have
started to evaluate growth kinetics of bacterial pathogens in mixed
cultures with spoilage bacteria such as pseudomonads and lactic acid
bacteria (LAB) (8, 9, 16, 18). In addition, specific
models have been proposed, designed, evaluated or revalidated to
predict growth of mixed microbial populations in meat and other foods
(1, 7, 28, 49, 51). This may indicate an increasing scientific interest in the effects of microbial competition on food
preservation and safety.
Meat decontamination technologies, including spraying or rinsing of
animal carcasses before evisceration and/or before chilling with cold
or hot water or chemical solutions (e.g., organic acids), or exposure
to steam, are used extensively in the United States to reduce microbial
contamination in meat (15, 44, 45, 48). These
interventions are integrated into meat safety management systems, such
as hazard analysis critical control point, which is required by
regulation in the United States (21, 46), and are proposed
to be used in meat cuts before packaging and shipment to the market
(39, 45, 48). Despite their established effectiveness in
reducing carcass surface contaminants (by approximately 1 to 3 logs),
the residual efficacy of decontamination interventions and their
residual effects on the natural flora of the meat and the plant
environment have not been established adequately (44, 45,
48). The potential development of stressed bacterial pathogens that may cross-contaminate and multiply on meat or colonize meat processing plants are important safety issues that have yet to be
addressed (45, 48). The risk of creating stressed
pathogens may be greater following organic acid decontamination due to
the residual effect of acids on the meat or in the waste and the longer adaptation times of pathogens to acid, especially if microbial niches
are established in the plant environment.
The impact of pathogen reduction strategies on microbial ecology of
meat plant environments and resulting products and the relative safety
of products with low compared to high numbers of background flora has
been questioned (26). It has been suggested that a
possible explanation for the increasing number of foodborne outbreaks
in the United States is that food may now have low numbers of
antagonists to suppress pathogen survival and growth, and that could be
due to decontamination (25, 26). Indeed, high levels of
natural flora were recently shown to inhibit growth of
Escherichia coli O157:H7 in ground beef (50).
However, to our knowledge, limited data exist on the interactions
between the natural flora and responses of pathogenic bacteria to
stresses (2, 17), even though it seems logical that such
interactions should occur in food environments (3, 22,
47).
The objective of this study was to investigate the effect of the
natural flora on the acid tolerance response (ATR) of Listeria monocytogenes in a model system simulating waste fluids that may be present in a fresh beef processing plant. Following inoculation (105 CFU/ml) of the pathogen in fresh beef decontamination
fluids (washings) with or without prior filter sterilization, bacterial growth was monitored at 35°C while the ATR was evaluated by periodic exposure of L. monocytogenes cells grown in the absence or
presence of background flora to acid. High (35°C) incubation rather
than lower (15°C) commercial temperatures were selected in order to maximize the growth potential and ATR of L. monocytogenes in
presence versus absence of a competitive natural flora. Although
E. coli O157:H7 and Salmonella are the main
target organisms of meat decontamination (44, 48),
L. monocytogenes was selected because of its high incidence
on fresh meat and poultry (41, 47), its prevalence in meat
processing plants (41), its potential to survive meat decontamination when present as a natural contaminant
(39), and its resistance to multiple stresses and superior
ability to form biofilms (30, 31).
(Part of this work was presented as poster P-055 at the 87th Annual
Meeting of International Association for Food Protection, August 6 to
9, 2000, Atlanta, Ga.)
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MATERIALS AND METHODS |
Bacterial strains.
L. monocytogenes N-7155
(serotype 1/2b) isolated from meat (6) was used throughout
this study. In addition, a streptomycin (800 µg/ml)-resistant
derivative of another meat isolate (6) of L. monocytogenes, N-7144 (serotype 1/2b, N-7144Sm+), developed in
previous studies (54) was used in some experiments to
selectively detect the pathogen on all-purpose media in order to
support findings on selective media. Selection of strains was based on
their meat origin and increased acid tolerance compared to other
L. monocytogenes strains tested, including strain Scott A
commonly used in food research, as will be described later. Strains
were available as frozen (
70°C) stock cultures in Trypticase soy
broth (BBL, Becton Dickinson Co., Cockeysville, Md.) with 0.6% yeast
extract (Difco, Becton Dickinson Co., Sparks, Md.) (TSBYE),
supplemented with 20% glycerol. Strains were activated by transferring
0.05 ml of stock inoculum in 10 ml of TSBYE followed by overnight
incubation at 35°C. Working cultures were kept on Trypticase soy agar
(BBL) with 0.6% yeast extract (TSAYE) slants at 4°C and transferred monthly. Strains were subcultured twice in TSBYE at 35°C for 24 h before use in experiments.
Preparation of fresh meat decontamination fluids (washings).
Fresh (72 h postmortem), nondecontaminated top rounds of beef were
obtained from a commercial plant or from the Meat Science Laboratory of
Colorado State University and used to prepare washings within 24 h
after transportation. Each top round was cut into four portions
weighing approximately 2 kg each. Each portion was individually spray
washed with 2 liter of one of the following solutions: (i) cold
(10°C) tap water, (ii) hot (85°C) tap water, (iii) a 2% warm
(55°C) solution of lactic acid (DL-lactic acid; 85%
[wt/wt]; Sigma, St. Louis, Mo.) in tap water, or (iv) a 2% warm
(55°C) solution of acetic acid (glacial acetic acid; 100%; Mallinckrodt Baker Inc., Paris, Ky.) in tap water. These
decontamination treatments were selected because they are commonly used
in meat plants in the United States. An additional reason for spraying meat with water at two different temperatures (10 or 85°C) was to
account for potentially lower populations of natural flora, and
potentially higher organic matter, in hot compared to cold water
washings, due to the greater bacterial killing and nutrient extracting
effect of 85°C spray water. Such potential differences might affect
subsequent growth of L. monocytogenes inoculated in the
washings after meat decontamination. Spraying of meat portions was done
with hand spray washers (Contico Int., St. Louis, Mo.). During
spraying, the nozzle of the spray washer was kept at approximately 20 cm from the meat surface, while it was moved slowly around the
suspended meat to ensure uniform spraying of all sides. A plastic bowl
was placed under the meat to collect the washings. The meat was allowed
to drain for 5 min, and then the washings were distributed in 500-ml
amounts in presterilized screw-capped bottles (Nalgene). The washings
were kept at 4°C if handled on the same day, or stored at 30°C
and used within 30 days, following overnight thawing at 4°C. For use
in these experiments, each type of washing was divided in two parts.
One part was inoculated with L. monocytogenes without any
treatment (unfiltered washings), as described later, while the other
part was filtered through Whatman no. 1 filter paper using a Buchner
funnel under vacuum to remove large meat particles, and then each was
centrifuged at 12,000 rpm (Beckman model J2-21 centrifuge) for 30 min
at 4°C to remove small particles. The supernatant was filter
(0.2-µm pore size; Nalgene) sterilized under vacuum.
After preparation, the uninoculated meat washings were tested
microbiologically, as described below, to ensure sterility of the
filter-sterilized washings or to determine the numbers of natural meat
flora and any naturally occurring Listeria spp. in unfiltered washings. Also, the pH of the washings was measured before
storage and during the experiments. A digital pH meter (Accumet 50;
Fisher Scientific, Houston, Tex.) with a glass pH electrode (Hanna
Instruments, Ann Arbor, Mich.) was used for measurement of pH.
Culturing of L. monocytogenes in the washings.
Portions (100 ml) of each type of filter-sterilized or unfiltered
washings were distributed in 250-ml presterilized bottles (Nalgene) and
inoculated with stationary-phase cultures (TSBYE at 35°C for 24 h) of L. monocytogenes N-7155 or N-7144Sm+ (approximately 105 cells/ml). Treatments, including uninoculated (control)
washings of meat, were incubated statically at 35°C for 8 days.
Samples were taken for microbiological analysis and pH measurement at 0, 1, 4, and 8 days of incubation. Serial decimal dilutions in 0.1%
buffered peptone water (Difco) were prepared and then plated in
duplicate on TSAYE and PALCAM (Difco) agar plates to determine total
bacterial counts and populations of inoculated L. monocytogenes, respectively. When strain N-7144Sm+ was inoculated
in unfiltered washings, TSAYE supplemented with 800 µg of
streptomycin sulfate (Sigma) per ml (TSAYE-Sm) was used, in addition to
PALCAM, to selectively enumerate L. monocytogenes. Colonies
on agar plates were counted after incubation at 35°C for 48 h.
For counts below the analysis detection limit (<1.0 log CFU/ml), 5 ml
of culture was added to 45 ml of Listeria enrichment broth
(Difco) and incubated at 35°C for 48 h for enrichment. Portions
(0.1 ml) of the enriched culture were spread in duplicate on PALCAM
plates and incubated at 35°C for 48 h, after which plates were
checked for Listeria growth.
Ten percent of colonies on countable TSAYE plates were tested for Gram
reaction by mixing colonies with a 3% KOH solution
to observe presence
(gram negative) or absence (gram positive)
of a slimy suspension.
Representative gram-negative colonies (three
to five) were subjected to
the oxidase test by using an Oxy-Swab
(Remel Inc., Lenexa, Kans.) or
dry slide (BBL) kit. All gram-positive
colonies on plates were
subjected to the catalase test by dropping
a 3%
H
2O
2 solution directly onto them to observe
effervescence.
The microscopic appearance of representative colonies
was checked
in wet mount or Gram-stained
cultures.
Assessment of acid tolerance.
As mentioned, acid challenge
experiments were performed to select strains N-7155 and N-7144Sm+ as
the most acid tolerant among other L. monocytogenes strains
available in our laboratory and to assess the acid tolerance of
stationary-phase cells used to inoculate meat washings (day 0).
Screening included strain Scott A, as well as the parental strain
N-7144 to ensure that resistance to streptomycin did not alter the acid
tolerance of the Sm+ derivative. These experiments were also useful in
the selection of the most appropriate challenge media for later use, as
discussed in Results. More specifically, the screening was conducted
with TSBYE acidified to pH 3.5 or 2.5 with lactic or acetic acid.
Lactic and acetic acids were selected as acidulants because of their
commercial application for meat decontamination. Selection of challenge
pH 3.5 or 2.5 was based on the pH range of organic acid solutions applied in meat decontamination. It was also based on the pH of most
challenge media reported in the literature for L. monocytogenes and other food pathogens (14, 29, 30, 31,
52), and for L. monocytogenes with lactic acid
(TSBYE, pH 3.5) in particular (37). The organic acid
reagents used for preparation of the challenge media (the same as those
used to prepare the 2% acid solutions for meat decontamination) were
added to TSBYE (pH 7.2) undiluted before distribution in tubes and
autoclaving. Challenge media were checked after sterilization to ensure
that their pH was within 0.05 unit of the desired value.
Following growth of
L. monocytogenes strains in 10 ml of
TSBYE for 24 h at 35°C, 1 ml containing approximately
10
6 cells suspended in 0.1% buffered peptone water of each
strain
was pipetted into 9 ml of TSBYE acidified to pH 3.5 or 2.5 with
lactic or acetic acid. This gave a concentration of approximately
10
5 cells/ml exposed to acid (i.e., the inoculation level
in meat
washings). Addition of cells to TSBYE with no pH adjustment (pH
7.2) served as a control. During acid challenging, tubes were
kept at
25°C. Samples (1 ml) were periodically (0, 30, 60, 90,
and 120 min)
taken from each tube, serially diluted in 9 ml of
0.1% buffered
peptone water, and plated on TSAYE or PALCAM plates
to determine the
number of survivors and potential differences
in recovery on PALCAM due
to acid injury. Strain N-7144Sm+ was
also plated on TSAYE-Sm to
evaluate the effect of the antibiotic
on pathogen recovery. Colonies on
plates were counted after incubation
at 35°C for 48 h. To
account for potentially higher populations
of
L. monocytogenes grown in the washings and then acid challenged,
the
selected strains were also exposed (10
8 CFU/ml) to TSBYE
acidified (pH 3.5) with lactic acid and to unfiltered
2% lactic acid
(pH 2.5) and 2% acetic acid (pH 3.2) washings of
meat. The acid
solution washings were used as challenge media
since bacterial
pathogens may be transferred to such waste fluids
following
decontamination of meat. Survivors were determined as
described
above.
In experiments with meat washings, the ATR of inoculated
L. monocytogenes was comparatively determined in the absence (i.e.,
filter-sterilized washings) or presence (i.e., unfiltered washings)
of
natural flora after 1 and 8 days of storage at 35°C. The acid
challenge procedure was as described above. One milliliter of
each
washing culture was pipetted into each of four tubes containing
9 ml of
a selected acid challenge medium. Selected challenge media
were TSBYE
adjusted to pH 3.5 with lactic acid, unfiltered 2%
lactic acid (pH
2.5) or 2% acetic acid (pH 3.2) washings, and
TSBYE (pH 7.2), which
served as a control. TSBYE adjusted to pH
3.5 with acetic acid, or to
pH 2.5 with either lactic or acetic
acid, was not used, for reasons
presented in Results. Portions
(1 ml) were taken from each challenge
medium at 0, 30, 60, 90,
and 120 min after inoculation and analyzed on
TSAYE and PALCAM,
as well as on TSAYE-Sm when strain N-7144Sm+ was
used, as for
pure cultures. Colonies (10%) on TSAYE plates from
unfiltered
samples were tentatively characterized as described above to
determine
the overall composition of the natural flora which occurred
in
coculture with
L. monocytogenes and survived acid
challenging.
Especially for presumptive colonies of LAB (i.e., white,
gram
positive, catalase negative), representative cultures were
isolated
in MRS broth and then tested for growth on
Enterococcus agar (Difco).
All experiments were done in
triplicate, by inoculating either
different batches of washings with
strain N-7155 or the same batch
of washings with strains N-7155 and
N-7144Sm+. Microbiological
counts were converted to log CFU per
milliliter, and the means
and standard deviations (SD) were
calculated.
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RESULTS AND DISCUSSION |
Microbial contamination of meat washings.
Cold (10°C) and
hot (85°C) water meat washing fluids had average total microbial
(TSAYE) counts after preparation of 3.9 ± 0.6 and 4.9 ± 0.6 log CFU/ml, respectively. The higher natural flora counts of hot
compared to cold water washings may have been due to hot water having
removed more bacteria in the washings without necessarily killing them.
Filter-sterilized water washings were found to be free of background
flora, and when incubated (35°C, 8 days) without inoculation, no
microbial growth (<1.0 log CFU/ml) occurred (data not shown). The 2%
lactate and 2% acetate solution unfiltered and uninoculated washings
contained natural flora of <1.0 log CFU/ml, and no microbial growth
(<1.0 log CFU/ml) occurred during storage at 35°C. Thus, the
microbial populations of inoculated filter-sterilized water washings or
filter-sterilized or unfiltered acid solution washings developing on
TSAYE were only L. monocytogenes, unlike those of unfiltered
water washings, in which the changes of the natural flora were followed
in coculture with the inoculated strains.
Behavior of L. monocytogenes in meat washings with or
without natural flora.
Inoculated L. monocytogenes died
off (<1.0 log CFU/ml upon direct plating on PALCAM or TSAYE) within 24 h in all acid solution washings at 35°C, irrespective of acidulant
present or filter sterilization (Table
1). In all experiments, the complete
inactivation of the pathogen in acid solution washings was confirmed by
negative culture enrichment (data not shown), which was done in
parallel with direct plating on PALCAM to verify absence of the
pathogen when counts were lower than 1.0 log CFU/ml. Additional new
data from our laboratory (43) have confirmed that the
35°C incubation temperature had an accelerating effect on the
pathogen inactivation rate in acid solution washings compared to lower
(4 or 10°C) incubation temperatures. At 4 and 10°C, L. monocytogenes may survive for 2 and 4 days in 2% lactate or 2%
acetate washings, respectively, while E. coli O157:H7 may
survive longer than L. monocytogenes, as the former pathogen
was countable after 7 days in 2% acetate washings stored at 4°C
(43). Thus, compared to incubation at the optimum (35°C)
growth temperature, purposefully used in this study, the generally
lower temperatures encountered in meat plants may enhance survival of
acid-adapted pathogens, which may serve as potential
cross-contamination sources, if pathogen niches are established in the
plant (43). It should be noted, however, that spots near
motor and conveyor belts or other equipment in meat plants may reach
temperatures higher than those found in most other areas of a plant.
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TABLE 1.
Changes in populations of natural flora (TSAYE) and
inoculated L. monocytogenesa (PALCAM) in fresh
beef decontamination fluids (spray-washings) stored at 35°C with
or without previous filter sterilizationa
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In nonacid (water) washings,
L. monocytogenes populations
increased by approximately 0.9 to 1.8 log CFU/ml by day 1, irrespective
of natural flora (Table
1). Then, pathogen numbers remained constant
in
unfiltered water washings or declined by approximately 1.0
log in
filter-sterilized water washings. This decline may indicate
that
filter-sterilized water washings contained fewer nutrients
than
unfiltered washings following centrifugation and filtration.
Overall,
neither type (10 or 85°C) of water washings supported
extensive
pathogen growth, indicating that the nutrient requirements
of
L. monocytogenes may have been higher than the nutrient supply
in the
water washings of meat. This, however, was not the case
with the
natural microbial flora (TSAYE), which increased rapidly
(>10
8 CFU/ml), outgrew
L. monocytogenes by at
least 1 to 2 logs by
day 1, and remained at high numbers throughout
storage of unfiltered
water washings at 35°C (Table
1). The behavior
of the natural
flora in the unfiltered water washings without
inoculation with
L. monocytogenes was very similar to that
in the inoculated samples
(data not shown). Colonies of natural flora
on TSAYE plates of
water washings stored for 1 to 8 days at 35°C were
creamy or yellowish
gram negative, while plates were practically free
of
L. monocytogenes colonies (flat, whitish, with an erased
center). Preliminary characterization
of this flora indicated that it
was a mixture of oxidase-positive
(i.e.,
Pseudomonas or
related genera) and oxidase-negative (i.e.,
family
Enterobacteriaceae) bacteria, with the former type being
more numerous (60 to 75%) overall, especially in water (10°C)
washings (75 to 85%). Other data from our laboratory showed that
unfiltered water (10 to 85°C) meat washings inoculated with
L. monocytogenes, E. coli O157:H7, or
Salmonella and
stored at 4
or 10°C were similarly dominated by gram-negative flora,
which,
however, was shifted to >90% oxidase positive (i.e.,
Pseudomonas-like
bacteria) (
43). Thus, at
35°C, the composition of the natural
flora may have been more diverse
between water treatments or replicates
compared to 4 or 10°C
(
43), depending on the type of bacteria
present in the
water washings after spraying and the ability of
most
Enterobacteriaceae to compete better at high temperatures.
Further studies are needed to characterize this background flora
at the
genus and species level and to evaluate its potential influence
on
pathogen
behavior.
Several previous studies have shown that, depending on the food and its
storage conditions, the background flora, composed
mainly of
Pseudomonas or LAB may inhibit (
4,
10,
11,
35),
stimulate (
19,
33,
34), or have no effect (
5,
34) on
L. monocytogenes. Inhibition of
L. monocytogenes in mixed cultures
in broth was due to a lengthening
of the lag phase, slowing of
growth, and suppression of the maximum
population density (MPD)
(
8,
9). The magnitude of the
competitive or inhibitory effect
of the background flora may be
influenced by temperature, pH,
and nutrient depletion (
8,
9), while sometimes siderophores
and bacteriocins produced by
Pseudomonas (
11) and LAB (
24),
respectively, may also enhance suppression of
L. monocytogenes.
In this study, there was no direct evidence that
the background
flora inhibited or stimulated growth of
L. monocytogenes in unfiltered
washings of meat at 35°C. In fact,
L. monocytogenes growth seemed
to be unaffected by the
predominant gram-negative flora, as the
MPD of the pathogen by day 1, in the presence or absence of competitors
in water washings, was
virtually the same (Table
1). However,
the rapid and high (>8 logs)
establishment of this type of natural
flora in the washings during
storage at 35°C appeared to induce
important changes at the cellular
level in
L. monocytogenes that
modulated the magnitude and
timing of expression of ATR, as discussed
below.
Effect of natural flora and L. monocytogenes on the pH
of washings.
The initial pHs of the water washings were similar
(6.0 to 6.1), regardless of filter sterilization or whether cold
(10°C) or hot (85°C) water was used to generate them (Table
2). During storage at 35°C, however,
major differences in pH were observed, which were dependent on the
presence or absence of natural flora and inoculated L. monocytogenes. Following the increases (>108 CFU/ml)
in natural flora of the unfiltered water washings, their pH increased
on average from 6.0 to 6.1 at inoculation (day 0) to 6.9 to 7.7 by day
8, irrespective of pathogen presence (Table 2). Interestingly, though,
the pH of washings inoculated with L. monocytogenes was
reduced 0.3 to 0.5 unit by day 1, while overall, this pH decrease did
not occur in the corresponding uninoculated samples (Table 2).
Probably, following its 5-log inoculation and approximate 1 to 2-log
growth at 35°C within 24 h, L. monocytogenes may have
utilized via fermentative pathways most of the available glucose in the
washings (16) to cause the observed pH reduction. Since
similar pH reductions were rare in unfiltered but uninoculated washings, it seems that the dominating gram-negative flora used alternative metabolic pathways to support its growth (13).
Indeed, in all cases there was a pH reduction in the washings by day 1, which was followed by a shift toward high pH values and development of
putrid off odors at later days of incubation at 35°C (i.e., as soon
as by-products of the gram-negative flora accumulated) (Table 2). In
our other study (43), L. monocytogenes
inoculated in unfiltered water (10 or 85°C) washings at
105 cells/ml increased by 0.6 to 1.3 logs at 4 or 10°C
but caused no reductions in pH. These findings suggest that L. monocytogenes maximized its growth and competition for glucose
uptake at 35°C (Table 1), while at 4 or 10°C (43) it
grew more slowly than the better-adapted meat spoilage flora, which
through its higher metabolic activity counteracted pH reductions in the
washings. In accordance, the pH of inoculated filter-sterilized water
washings was also reduced 0.4 to 0.7 unit at day 1, as a result of
L. monocytogenes growth, and finally reached values of
approximately 5.0 (Table 2), evidently because there was no natural
flora to counteract the pH reduction through its own metabolic
activity. Also, the pH of filter-sterilized but uninoculated washings
did not change during storage (Table 2), confirming the observation
that pH reductions in the inoculated samples were due to L. monocytogenes growth. Meanwhile, the pH of acid solution washings
was practically unchanged during storage, irrespective of filter
sterilization or pathogen inoculation (Table 2), reflecting the absence
of natural flora and the rapid inactivation of the inoculated pathogen at 35°C (Table 1).
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TABLE 2.
Changes in pH of fresh beef decontamination fluids (spray
washings) inoculated with L. monocytogenesa
with or without previous filter sterilization and stored at 35°C
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Acid tolerance of L. monocytogenes inoculum.
Stationary-phase (grown in TSBYE at 35°C for 24 h) cells of
L. monocytogenes were resistant to acid, but not under all
conditions tested. While TSBYE acidified to pH 2.5 with
lactic or acetic acid, or to pH 3.5 with acetic acid, completely
inactivated (<1.0 log CFU/ml) all strains tested within 0 to 120 min
(data not shown), most strains displayed a profound survival in
TSBYE acidified to pH 3.5 with lactic acid. Specifically, the
population reductions on TSAYE of strains N-7155,
N-7144Sm+, and N-7144 after 2-h exposure to lactic acid (pH
3.5) were on average 1.2, 1.2, and 1.1 logs, respectively (Fig.
1A). The respective reductions on
PALCAM were 3.2, 2.9, and 2.9 logs, indicating extensive acid
injury. The survival of strain N-7144Sm+ on TSAYE-Sm,
however, was similar to that on TSAYE (Fig. 1A), indicating
that streptomycin was not inhibitory to the acid-injured cells. The
acid tolerance of strains N-7155 and N-7144 was remarkably higher than
that of strain Scott A (Fig. 1A) and other L. monocytogenes
strains (data not shown), while that of strain N-7144Sm+ was similar to
that of its parental N-7144 strain (Fig. 1A); thus, these strains were
selected for further study. Notably, these findings suggest that
strain Scott A, which is commonly used in food research
(14, 30, 31, 53), may not be the most suitable
for studies dealing with L. monocytogenes survival in acid
foods. When 108 TSBYE cells (35°C, 24 h)
of strain N-7155 were exposed to TSBYE acidified (pH 3.5)
with lactic acid and to the unfiltered 2% acetic (pH 3.2) or 2%
lactic acid washings, the pathogen survived much better in the former
two challenge media (Fig. 1B). Strain N-7144Sm+ showed similar
results (data not shown).
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FIG. 1.
(A) Comparative survival of L. monocytogenes
N-7155, N-7144, N-7144Sm+n and Scott A on
TSAYE, PALCAM, and TSAYESm (strain
N-7144Sm+ only) after exposure to TSBYE acidified with lactic
acid (pH 3.5). Cells were challenged to acid after growth as pure
cultures in TSBYE at 35°C for 24 h. Cell numbers
exposed to acid at time zero were determined in TSBYE (pH
7.2, control), where no population reductions occurred within 120 min
(data not shown for graph clarity). Values are the mean of three
replicates (SD range, 0.1 to 0.5). (B) Comparative survival of high
levels (108 CFU/ml) of L. monocytogenes
N-7155 on TSAYE or PALCAM after exposure to
TSBYE acidified with lactic acid (pH 3.5) and to unfiltered
2% acetate (pH 3.2) or 2% lactate (pH 2.5) spray washings (AA-SW or
LA-SW) of meat. Cells were challenged to acid after growth as pure
cultures in TSBYE at 35°C for 24 h. Cell numbers
exposed to acid at time zero were determined as above. Values are from
one representative experiment.
|
|
These findings confirm the major differences in the buffering capacity
of the challenge media used and suggest differences
in the acidifying
capacity and bactericidal mode of action of
each acid, which depend on
both the acid ionization constant (solution
pH) and concentration in
solution (
53). Indeed, TSBYE (100 ml)
required
an average addition of 1.3 or 8.6 and 4.7 or 42.8 ml
of lactic or
acetic acid, respectively, to adjust the pH to 3.5
and 2.5, respectively. Conversely, 100 ml of 2% (wt/vol) acidic
meat washings
containing approximately 2 ml of each of lactic
(85% [wt/wt];
density, 1.2) or acetic (100% glacial) displayed
a pH of 2.5 or 3.2 after spraying, respectively. These results
confirm that acetate is a
weaker acid than lactate and that TSBYE
has a higher
buffering capacity compared to meat washings. TSBYE
acidified
to pH 2.5 with acetic acid was of immediate lethality
(<1.0 log CFU/ml
at time zero on all recovery media) to all
L. monocytogenes
strains, while TSBYE acidified (pH 2.5) with lactic
acid
resulted in no survivors (<1.0 log CFU/ml) at 60 min (data
not shown).
In contrast, the 2% lactate washings of pH 2.5 permitted
survival for
30 to 60 min (Fig.
1B). Accordingly, TSBYE acidified
to pH
3.5 with acetate was more lethal to
L. monocytogenes than
2% acetate washings, although of higher pH. Thus, the concentration
of
acetic acid rather than its pH in solution was more decisive
for
L. monocytogenes inactivation, a result consistent with
evidence
that acetate kills the pathogen primarily by lowering the
intracellular
pH following its penetration through the membrane and
dissociation
inside the cell (
53). In contrast, the 2%
lactate washings (pH
2.5) were consistently more lethal to
L. monocytogenes than acidified
TSBYE (pH 3.5) (Fig.
1B),
having a higher acid concentration and
lower pH in solution. In
conclusion, the stationary cells of strains
N-7155 and N-7144Sm+ were
acid tolerant at inoculation of meat
washings (day 0), while
TSBYE acidified (pH 3.5) with lactic acid
and the original
2% lactate (pH 2.5) or acetate (pH 3.2) solution
washings were the
most suitable challenge media for use in the
experiments.
Effect of natural flora on ATR of L. monocytogenes in
water washings.
The acid tolerance of L. monocytogenes
was evaluated in unfiltered or filter-sterilized inoculated water (10 or 85°C) washings but not in acid solution washings because the
latter did not allow survival of the inoculum at day 1 or 8 at
35°C. Changes in ATR of the pathogen in the water washings during
storage were dependent on the presence or absence of natural flora
and associated pH changes. In filter-sterilized water (10 or 85°C)
washings, L. monocytogenes N-7155 (PALCAM)
increased its acid tolerance by 1.0 to 2.9 logs from days 1 to 8 (Fig.
2). Consistent with previous findings
(Fig. 1B), the pathogen survived better in acidified TSBYE
(pH 3.5) with lactic acid and 2% acetate washings (pH 3.2) than in 2%
lactate washings (pH 2.5) and better on TSAYE (Fig. 3) than on PALCAM (Fig. 2).
Importantly though, following its preincubation and growth in the water
washings, the recovery of strain N-7155 on PALCAM (Fig. 2)
was slightly lower than that on TSAYE (Fig. 3), which was not
the case in the cells from TSBYE cultures used as inocula
(Fig. 1A). This finding may indicate that the oligotrophic environment
of the washings enhanced the acid tolerance of L. monocytogenes due to starvation (30, 31), and thus
the proportion of acid-injured cells that were unable to grow on
PALCAM to the total surviving populations was reduced.
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FIG. 2.
Acid survivors of L. monocytogenes N-7155
(PALCAM) after exposure to TSBYE acidified with
lactic acid (pH 3.5), 2% acetate solution spray washings of meat (pH
3.2) (AA-SW), or 2% lactate solution spray washings of meat (pH 2.5)
(LA-SW). Cells were challenged as for Fig. 1 after 1 or 8 days of
growth in filter-sterilized spray washings of meat previously
decontaminated with cold (10°C; A) or hot (85°C; B) water. Cell
numbers exposed to acid at time zero were determined as for Fig. 1.
Values are the mean of three replicates (SD range, 0.1 to 0.8).
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FIG. 3.
Acid survivors of L. monocytogenes N-7155
(TSAYE) after exposure to TSBYE acidified with
lactic acid (pH 3.5), 2% acetate solution spray washings of meat (pH
3.2) (AA-SW), or 2% lactate solution spray washings of meat (pH 2.5)
(LA-SW). Cells were challenged as for Fig. 1 after 1 or 8 days of
growth in filter-sterilized washings of meat previously decontaminated
with cold (10°C; A) or hot (85°C; B) water. Cell numbers exposed to
acid at time zero were determined as for Fig. 1. Values are the mean of
three replicates (SD range, 0.1 to 0.9).
|
|
An opposite trend was observed for
L. monocytogenes grown in
unfiltered water washings, where the pathogen (PALCAM) was
appreciably
more acid tolerant by day 1 than by day 8 (Fig.
4). Specifically,
when
L. monocytogenes was exposed to acidified TSBYE (pH 3.5)
with lactic acid and 2% acetate washings (pH 3.2) at day 1, its
average population reductions ranged from 0.3 to 1.4 logs. The
respective reductions at day 8 ranged from 3.0 to >5.0 log CFU/ml
(Fig.
4). These results indicated that
L. monocytogenes was
sensitized
to acid by approximately 1.5 to 4.5 logs from days 1 to 8 at
35°C
in the presence of natural flora. Notably, sensitization was
greater
with acetic acid at pH 3.2 (Fig.
4), which was not the case
either
with the inoculum (Fig.
1B) or with stationary cells in
filter-sterilized
washings by day 8 (Fig.
2). Lactate (2%) washings
(pH 2.5) were
again the most lethal challenge medium (Fig.
4).
Inoculation of
the same batch of unfiltered washings with strain
N-7144Sm+ indicated
that acid sensitization of
L. monocytogenes was neither strain
dependent nor overestimated due
to potentially low recovery on
PALCAM of viable but
acid-injured cells (Fig.
5). Indeed,
although
populations on PALCAM were constantly lower than
those on TSAYE-Sm,
the trend of
L. monocytogenes
N-7144Sm+ to become acid sensitive
following an 8-day exposure to
unfiltered water washings was unchanged
(Fig.
5).
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FIG. 4.
Acid survivors of L. monocytogenes
(PALCAM) after exposure to TSBYE acidified with
lactic acid (pH 3.5), 2% acetate solution spray washings of meat (pH
3.2) (AA-SW), or 2% lactate solution spray washings of meat (pH 2.5)
(LA-SW). Cells were challenged as for Fig. 1 after 1 or 8 days of
growth in unfiltered spray washings of meat previously decontaminated
with cold (10°C; A) or hot (85°C; B) water. Cell numbers exposed to
acid at time zero were determined as for Fig. 1. Values are the mean of
three replicates (SD range, 0.1 to 1.5).
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FIG. 5.
Comparative survival of L. monocytogenes
N-7144Sm+ on TSAYE-Sm (solid lines) or PALCAM
(dotted lines) after exposure to TSBYE acidified with lactic
acid (pH 3.5), 2% acetate solution spray washings of meat (pH 3.2)
(AA-SW) or 2% lactate solution spray washings of meat (pH 2.5)
(LA-SW). Cells were challenged as for Fig. 1 after 1 or 8 days of
growth in unfiltered spray washings of meat previously decontaminated
with cold (10°C; A) or hot (85°C; B) water. Cell numbers exposed to
acid at time zero were determined as for Fig. 1A. Values are from one
representative experiment.
|
|
The predominant, gram-negative flora of the water washings was very
sensitive to acid (Fig.
6), a result in
agreement with
previous data (
23). At day 1, colonies that
survived on TSAYE
plates after 120 min of exposure to acid
(Fig.
6) were mainly
L. monocytogenes and were slightly
more numerous than colonies
on PALCAM (Fig.
4). At day 8, however, following acid sensitization
of
L. monocytogenes
(Fig.
4 and
5), numerous (10
5 CFU/ml) white colonies other
than those of the pathogen developed
on TSAYE plates from all
samples (Fig.
6). Evidently, these indigenous
microorganisms increased
in relatively high numbers during storage
and, despite neutral pH
conditions in the unfiltered washings
by day 8 (Table
2), maintained a
high ATR and survived even in
2% lactate washings (pH 2.5) throughout
the acid-challenging period
(Fig.
6). Based on preliminary
characterization tests, these organisms
were LAB because they
were gram-positive, catalase-negative nonsporeformers
and were able to
grow and acidify (pH <5.0; 30°C, 24 h) MRS broth.
The profound
acid resistance of LAB compared to food spoilage
gram-negative bacteria
is well established (
38). More than 80%
of those isolates
were cocci but not enterococci, because they
did not grow on
Enterococcus agar. Additional tests are required
to better
characterize these isolates and evaluate their influence
on the
behavior of
L. monocytogenes under these conditions.
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FIG. 6.
Acid survivors of natural flora (TSAYE) after
exposure to TSBYE acidified with lactic acid (pH 3.5), 2%
acetate solution spray washings of meat (pH 3.2) (AA-SW), or 2%
lactate solution spray-washings of meat (pH 2.5) (LA-SW). Cells were
challenged as for Fig. 1 after 1 or 8 days of growth in unfiltered
spray washings of meat previously decontaminated with cold (10°C; A)
or hot (85°C; B) water. Cell numbers exposed to acid at time zero
were determined as for Fig. 1A. Values are the mean of three replicates
(SD range, 0.1 to 1.8).
|
|
This study evaluated the potential of the natural flora to modulate the
ATR of
L. monocytogenes in a food environment. The
results
have potential implications for meat plant environments
where the
pathogen may be found in association with natural microbial
flora in
residues of meat spray-washing solutions. Establishment
of such niches
in a meat plant may have potential implications
for food safety through
product cross-contamination. Few studies
have attempted to correlate
pathogen response to stresses with
the natural flora in foods. Duffy et
al. (
17) reported that
the addition of 10
8 CFU
of viable competitors per ml protected an underlying, exponentially
growing population (10
5 CFU/ml) of
Salmonella
serovar Typhimurium against thermal inactivation,
as the pathogen
D values at 55°C increased from 0.43 to 2.09.
Furthermore, Aldsworth et al. (
2) showed that the
presence
of 10
8, but not 10
5 to
10
7, CFU of mixed viable competitors per ml also protected
Salmonella serovar Typhimurium (10
5 CFU/ml) from
freezing, but addition of equal populations of heat-killed
competitors
did not provide any protective effect. Thus, the observed
protective
effect to food-related stresses, such as heat and freezing,
was
associated with the metabolic activity of the competitive
flora; most
importantly, it did not correlate with the stationary-phase
stress
adaptation, because it was essentially instantaneous after
mixing of
Salmonella serovar Typhimurium cells with the competitors
(
2). It was also found that by rapidly reducing the levels
of dissolved oxygen through active respiration, high levels of
competitive flora reduced oxidative damage to exponential-phase
cells
of
Salmonella serovar Typhimurium and decreased the RpoS
induction time, thus arresting pathogen growth and conferring
protection from stress (
2). On this basis, a novel
hypothesis
has been advanced to explain the increased sensitivity to
stress
of exponential-phase compared to stationary-phase bacterial
cultures,
the suicide response (
2,
3). According to this
hypothesis,
pure cultures of rapidly growing and respiring bacterial
cells
exposed to mild stresses will suffer growth arrest but their
metabolism
will continue to result in a burst of free-radical
production
that is lethal to the cells, rather than the stress itself
(
3).
This condition compares favorably with another recent
study (
18)
showing that
E. coli O157:H7 was
inhibited to a lesser extent
in coculture with a competitive flora than
in pure culture in
a simulated fermentation broth (pH 5.8 to 4.8) at
37°C. The authors
also suggested that the increased numbers of the
competitive flora
protected the pathogen from acid (
18);
however, the ATR of
E. coli O157:H7 in the absence versus
the presence of competitors
was not examined. Furthermore, none of the
above studies investigated
whether the protective effect of the
competitive flora on bacterial
pathogens will continue to exist upon
extended periods of coexistence
of growth-arrested pathogenic cells
with high competitor populations.
This is a condition that may apply in
meat environments, where
mixed microbial associations may be present
(
1,
5,
13,
27,
32,
35,
40,
43,
47).
In this study,
L. monocytogenes developed its acid tolerance
in filter-sterilized nonacid (water) meat washings in the same
manner,
but more slowly than in pure broth culture (
14,
37).
By
day 1 at 35°C, the pathogen in the oligotrophic environment
of these
washings seemed to be in a slow exponential phase and
was thus of
increased acid sensitivity. By day 8, however, the
pathogen had entered
into the stationary phase in filter-sterilized
washings. Thus,
surviving cells, although lower in numbers than
at day 1 (Fig.
2 and
3), were of higher acid resistance, probably
due to adaptation to
starving and low pH (5.0 to 5.3) conditions
(
30) as well
as to their stationary-phase-induced acid resistance,
which is
regulated by RpoS and is pH independent (
14).
The reversal of ATR of
L. monocytogenes in unfiltered water
washings at 35°C (Fig.
4 and
5) may be an important observation.
The
pathogen was very acid tolerant at day 1, indicating that
cells were
protected to acid earlier than those in filter-sterilized
washings,
even though there were no major differences in MPD of
the two cultures
by day 1 at 35°C (Table
1). In fact, the pathogen
at day 1 in the
unfiltered washings was more acid tolerant on
PALCAM (Fig.
4)
than it was the inoculum culture at day 0 on PALCAM,
or even
on TSAYE (Fig.
1A). Apart from the potential starvation,
this
accelerated ATR of
L. monocytogenes in unfiltered washings
appears to be in complete agreement with studies discussed (
2,
3,
17), indicating that high numbers of competitive flora
increase
resistance of bacterial pathogens to stress by arresting
growth and
accelerating entry into stationary phase. In agreement
with these
studies (
2,
17), the induction of the acid-protective
responses in
L. monocytogenes may have occurred soon after
the
competitive flora reached 10
8 CFU/ml in the washings
(i.e., within the first 24 h of incubation
at 35°C). However,
there are some remarkable differences between
the experiments of this
study and previous studies (
2,
17).
In our study, the
competitive flora was not mixed instantaneously
at high numbers (8 logs) with lower (5 logs) numbers of
L. monocytogenes in
broth, but it initiated growth from populations as low as 3
to 5 logs,
under competition of a 5-log
L. monocytogenes inoculum
in
the washings. Also, the mixed cultures were incubated statically
at
35°C, thus under reduced but not limited oxygen availability.
In
essence, although during the first incubation hours at 35°C
L. monocytogenes might have metabolized more actively, the rapidly
growing gram-negative flora would have steadily increased competition
for oxygen to support cell respiration. This may have shifted
the
metabolism of
L. monocytogenes, which is a typical
microaerophilic
organism though capable of increasing faster and more
extensively
under aeration (
31), to oxygen-independent
pathways (
16,
31).
Thus, the possibility that the
metabolic activity of
L. monocytogenes was accelerated to
compete against its natural competitors for
glucose uptake via
fermenting pathways cannot be ruled out, given
that experiments were
done near the optimum temperature (35°C)
of the pathogen
(
31). In other words, the lag phase of
L. monocytogenes might have been shortened and its exponential growth
might have
been accelerated, strictly depending on, and at the expense
of,
available glucose (
16) in the washings, while the
pathogen was
attempting to compete against its competitors. As
mentioned, this
may have caused the observed decrease in pH of
inoculated unfiltered
washings at day 1 at 35°C, which was uncommon
in the corresponding
samples without inoculation (Table
2). Probably,
L. monocytogenes converted some glucose to lactate
(
16), whereas, depending on
the proportional distribution
of pseudomonads or enterobacteria
in each occasion, the gram-negative
flora may have also converted
glucose to gluconate and oxygluconate
(
13) or lactate and acetate
(
13),
respectively, before they started to attack meat proteins
(
13,
47).
An important finding of this study appears to be the inability of
L. monocytogenes to maintain its high ATR (day 1), or
develop
a high stationary-phase acid resistance, in unfiltered nonacid
(water) washings at day 8 (Fig.
4 and
5). As mentioned, previous
studies did not address the potential changes in stress resistance
of
stationary-phase populations of bacterial pathogens underlying
much
higher populations of spoilage bacteria in foods or their
processing
plants for extended times. The observed acid sensitization
may have
been due to a shift in the intracellular activities and
catabolism of
L. monocytogenes to adapt and survive under the
conditions
created in the water washings by the gram-negative
spoilage flora. The
presence of meat particles in the washings
did not seem to offer any
protection for
L. monocytogenes to subsequent
acid exposure,
although such a protective effect has been seen
in enteric pathogens
(
52). Thus, the findings of this study
reveal that
L. monocytogenes may shift from acid resistant to
acid
sensitive following exposure in food environments of neutral
pH, such
as in nonacid (water) meat decontamination wastes. Research
is in
progress to determine if
L. monocytogenes and other
pathogens,
such as
E. coli O157:H7 and
Salmonella
serovar Typhimurium DT
104, are also sensitized to acid following
growth in pure compared
to mixed cultures with various spoilage
bacteria, and in poor
substrates compared to complex media, at
temperatures of 4 to
15°C to simulate more prevalent plant
conditions. Results have
shown that acid sensitization of bacterial
pathogens occurs following
exposure to water meat washings stored at
10°C, with
E. coli O157:H7
and
Salmonella
serovar Typhimurium DT 104 being sensitized more
than
L. monocytogenes, especially if previously acid adapted (J.
Samelis,
J. N. Sofos, P. A. Kendall, and G. C. Smith, unpublished
data).
The findings of this study may be of practical importance to the meat
industry. Since bacterial pathogens seem to be acid
sensitized under
the stressful conditions prevailing in nonacid
meat washings, the use
of water, steam, or other nonacid treatments
without or with reduced
use of acidic meat decontamination technologies
may be advantageous to
meat safety. This approach could potentially
minimize the risk of
establishment of acid-adapted pathogens in
the plant, while the
acid-sensitive pathogens surviving on decontaminated
meat or
transferred to nonacid wastes may be easily inactivated
in subsequent
processing steps. However, whether these potential
benefits of nonacid
decontamination technologies offset any potential
benefits due to
residual organic acid antimicrobial effects on
treated carcasses and
the resulting meat cuts (
44,
45) requires
further
research.
In summary, the present findings suggest that depending on its
composition, growth rate, and metabolic activity, the natural
microbial
flora of a food environment may protect or sensitize
pathogens to acid
stress and thereby affect food safety. Further
research is needed to
study competitive interactions of bacterial
pathogens with different
types of spoilage bacteria under various
sets of controlled
environmental conditions in order to better
understand and monitor
microbial competition as a component of
food safety
systems.
| |
ACKNOWLEDGMENTS |
Funding for this study was provided by USDA-CSREES (award
99-34382-8353) and by the Colorado Agricultural Experiment Station. John Samelis was a recipient of a NATO research grant from the Hellenic
Ministry of National Economy, Athens, Greece.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for Red
Meat Safety, Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523. Phone: (970) 491-7703. Fax: (970) 491-0278. E-mail: John.Sofos{at}colostate.edu.
| |
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0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.6.2410-2420.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
