1835

Journal of Food Protection, Vol. 70, No. 8, 2007, Pages 18351843

Copyright , International Association for Food Protection

Variation in Salmonella Resistance to Poultry Chemical

Decontaminants, Based on Serotype, Phage Type, and
Antibiotic Resistance Patterns
ROSA CAPITA*
Area de Nutricion y Bromatologa, Escuela Superior y Tecnica de Ingeniera Agraria, Avenida de Astorga, s/n. 24400-Ponferrada, Spain
MS 06-573: Received 8 November 2006/Accepted 14 February 2007

ABSTRACT
Chemical decontaminants are currently under review for final approval by the European Union authorities with the aim
of reducing the number and/or prevalence of pathogenic microorganisms on poultry. The purpose of the research being reported
here was to determine the association, if any, of decontaminant resistance with the serotype, phage type, and antibiotic
resistance of Salmonella strains. Sixty poultry isolates of Salmonella enterica (serotypes Enteritidis: phage types 1, 4, 4b, 6a,
14b, and 35; Typhimurium; Newport; Infantis; Poona; Virchow; Agona; Derby; and Paratyphi B) showing resistance to none
(sensitive), one (resistant), two, three, four, five, six, seven, or nine (multiresistant) antibiotics were screened for resistance to
1,000 ppm acidified sodium chlorite, 1.2% trisodium phosphate, or 25% citric acid. D-values (seconds required for 1-log
reduction in the number of bacteria) in peptone water, using a linear regression, of Salmonella in the presence of acidified
sodium chlorite varied widely with serotype (the highest resistance levels were shown by serotypes Typhimurium, Newport,
and Derby) and antibiotic resistance pattern (average values of 8.37 1.69 s for multiresistant strains as compared with 5.96
0.54 s for sensitive, P 0.05). A positive relationship (0.775, P 0.001) was found between acidified sodium chlorite
D-values and the number of antibiotics to which strains were resistant. Both serotype and antibiotic resistance had only a
slight influence over Salmonella resistance to trisodium phosphate, with average D-values from 12.44 0.91 s (sensitive
strains) to 13.28 0.77 s (multiresistant) (P 0.05). Neither serotype nor antibiotic profile was associated with Salmonella
resistance to citric acid (average D-value of 12.20 0.81 s). Minimal differences in resistance to decontaminants were found
among Salmonella Enteritidis phage types. Results in the present study highlight the importance of selecting an adequate strain
(serotype and antibiotic resistance pattern) when acidified sodium chlorite and trisodium phosphate are tested against Salmonella to ensure that concentrations capable of inactivating all strains are used.

Salmonella is among the commonest causes of human

enteric infections worldwide, and is responsible for considerable morbidity and some deaths. In Spain, salmonellosis
accounted for 80% of foodborne illness in the period 1993
to 2002 (18). Serotypes most frequently associated with human illness in this period were Enteritidis, Typhimurium,
Hadar, and Virchow (12). In addition to their pathogenicity,
there has been concern about antibiotic resistance in Salmonella strains, which has led to failures in treatments for
salmonellosis (26).
Live birds are the most important reservoir of Salmonella, and eggs and poultry are the foods that have most
commonly been implicated in such infections (31). In addition to good hygiene practices and hazard analysis and
critical control pointbased systems, the number and/or
prevalence of pathogenic microorganisms (e.g., Salmonella)
of fresh poultry and red meat can be reduced substantially
by the application of decontaminants.
At slaughtering plants in North America, it is normal
practice to subject carcasses to a variety of decontaminating
treatments during the carcass-dressing process to reduce microbial loads. Many of these interventions are approved by
* Author for correspondence. Tel: 34 987 442000; Fax: 34 987
442070; E-mail: rosa.capita@unileon.es.

the U.S. Food and Drug Administration (FDA) in poultry

processing plants as GRAS (generally recognized as safe)
substances, this being the case for 8 to 12% trisodium phosphate (TSP) or 1.5 to 2.5% organic acids, e.g., citric acid
(CA). Other chemicals are considered processing aids and
approved by the FDA as secondary direct food additives
permitted in food for human consumption, including sodium chlorite at 500 to 1,200 ppm combined with a GRAS
acid that achieves a pH between 2.3 and 2.9 in the solution
(5, 10, 25). However, in the European Union (EU) antimicrobials have not been permitted for treating poultry carcasses, parts, or viscera since 1971 (Directive 71/118/EC).
The EU meat hygiene regulations do not allow any method
or product decontamination other than washing with potable water or applying steam; regulators have argued that
processors would use antimicrobials to mask unhygienic
slaughtering or processing practices. However, Regulation
853/2004 of the European Parliament and of the Council,
laying down specific hygiene rules for food of animal origin
(1), applicable from 1 January 2006, provides a legal basis
to permit the use of a substance other than potable water
to remove surface contamination from products of animal
origin. In Annex II to the draft regulation, the Commission
introduced a provision aiming to authorize TSP, acidified
sodium chlorite (ASC), and chlorine dioxide as decontam-

1836

CAPITA

inants for poultry carcasses (SANCO 55/2005, revision 3,

Annex II). These chemicals are currently under review for
final approval by EU authorities. The European Food Safety
Authority panel on food additives, flavorings, processing
aids, and materials in contact with food has recently reported that poultry carcass decontamination with TSP, ASC,
chlorine dioxide or peroxyacid solutions, under FDA-approved conditions of use, poses no toxicological risk to human health (15). For these reasons, together with the favorable opinions of experts (30), it is expected that such
decontamination procedures will soon be authorized for
poultry carcasses in EU countries.
It has been suggested, however, that the decontamination processes could render the surface of the carcass susceptible to preferential growth of dangerous bacteria (e.g.,
some serotypes or antibiotic-resistant strains), because of
the removal of normal competitive microflora (7, 16). The
Federation of Veterinarians of Europe (16) recommends
that the decontamination of carcasses should not be allowed
unless it has been demonstrated that such techniques are
safe, taking into account the potential pathogenic microflora
involved.
In the last several years investigations have been carried out to determine the relative resistance to chemical
decontaminants of different spoilage and pathogenic microbial genera and species (8, 9). However, the variation in
resistance among strains of the same species has not so far
been studied. Intraspecies resistance variation is of importance, as care has to be taken to choose the appropriate
target strain for testing the effect of a decontamination procedure.
An association between resistance to antibiotics and
chemical biocides (other than poultry decontaminants) has
been previously demonstrated in Salmonella strains (3, 22,
27, 28). It has been hypothesized that some biocides and
antibiotics could share a common target or targets in bacteria cells (3). The Salmonella serotype characteristics have
also been shown to influence biocide resistance (2). However, chemical decontaminants for poultry have not so far
been tested for this purpose.
In order to determine the importance of choosing the
most appropriate target strain for use in poultry decontamination studies, the variation in Salmonella resistance to
three compounds (ASC, TSP, and CA) relative to serotypes,
phage types, and antibiotic resistance patterns has been determined for 60 poultry isolates.
MATERIALS AND METHODS
Microorganisms. A collection of 60 S. enterica strains, previously isolated from poultry in our laboratory, were used in this
study (Table 1). Isolates were maintained in tryptic soy agar (Oxoid, Ltd., Hampshire, England) slants at 3 1C until use.
The cultures for experiments were subcultured twice by inoculating into 10 ml of tryptic soy broth (Oxoid) supplemented
with 0.6% yeast extract (Oxoid) and incubated at 35C for 18 h
to achieve viable cell populations of 109 CFU/ml.
Preparation of chemical solutions. All glassware was thoroughly cleaned and then rinsed with distilled water and sterilized
before use. Stock solutions of ASC, TSP (Merck, Darmstadt, Ger-

J. Food Prot., Vol. 70, No. 8

TABLE 1. Characteristics of 60 isolates of Salmonella enterica

testeda
Serotype of
Salmonella

No. of antibiotics to
which strains are Antibiotic resistance No. of
resistant
pattern
strains

Enteritidis
Enteritidis PT 1
Enteritidis (PT 4)
Enteritidis (PT 35)
Enteritidis (PT 1)
Enteritidis
(PT 14b)
Enteritidis (PT 6a)

0
2
2
3
3
4

(sensitive)
(multiresistant)
(multiresistant)
(multiresistant)
(multiresistant)
(multiresistant)

Enteritidis (PT 1)

6 (multiresistant)

Enteritidis (PT 4)

7 (multiresistant)

Typhimurium
(URP)
Typhimurium
(URP)
Typhimurium
(DT 193)
Newport
Infantis
Infantis
Poona
Virchow (PT 8)
Agona
Derby
Paratyphi B

4
0
1
0
5
0
5
5

5 (multiresistant)

b
CF/C
S/SXT
EF/CP/TE
AMK/STR/K
S/SXT/TE/NF

21c
1
1
2
1
2
2

0 (sensitive)

AMP/AMC/EF/
CP/NA
S/SXT/EF/CP/NA/
TE
CZ/CF/S/SXT/EF/
CP/TE

2 (multiresistant)

EF/TE

9 (multiresistant)

STR/K/AMP/
AMC/S/SXT/T/
C/TE
GENS/S/SXT/TE

TE

S/SXT/EF/CP/TE

EF/CP/NA/TE/NF
AMP/AMC/S/
SXT/TE

(multiresistant)
(sensitive)
(resistant)
(sensitive)
(multiresistant)
(sensitive)
(multiresistant)
(multiresistant)

2
4
1

2
3
3
5
2
3
1
2

Iolates were screened for antimicrobial susceptibility using the

disk diffusion method, as described by NCCL standards (5, 22).
CF, cephalothin; C, chloramphenicol; S, sulfonamide; SXT, sulfamethoxazole-trimethoprim; EF, enrofloxacin; CP, ciprofloxacin; TE, tetracycline; AMK, amikacin; STR, streptomycin; K,
kanamycin; NF, nitrofurantoin; AMP, ampicillin; AMC, amoxicillinclavulanic acid; NA, nalidixic acid; CZ, cephazolin; T, trimethoprim; GEN, gentamicin; URP, unrecognized pattern.
b , no data.
c Isolates of phage types PT-1 (six strains), 4 (five), 4b (one
strain), and 14b (nine strains) were included.

many), and CA (Panreac, Barcelona, Spain) were aseptically prepared (wt/vol) at 0.11, 1.33, and 27.78%, respectively, in sterile
0.1% peptone water (Oxoid). For ASC preparation 0.11% sodium
chlorite (Fluka, Madrid, Spain) was acidified to pH 2.7 by adding
CA. All solutions were freshly prepared.
Inactivation procedure and enumeration of surviving
bacteria. Volumes of 18 ml of each solution were mixed at room
temperature (20 1C) in sterile tubes with 2 ml of bacterial
suspension (final concentration: about 5 108 CFU/ml). Different
tubes were used at different sampling times. After exposure of
Salmonella, 0.5 ml of 0.1 mol liter 1 sodium thiosulfate (SigmaAldrich Qumica, Madrid) was added into 20 ml of solution before
sampling to quench the activity of the biocides. Each sample from
the initial inocula and after chemical treatment was serially diluted
with sterile buffered peptone water (Oxoid) using 10-fold dilu-

5.59 0.15
(n 3)

5.96 0.54 AB
(n 33)

Salmonella
(all strains)
A

7.75 1.01
(n 3)

7.21 0.45 B a
(n 2)
8.81 0.99 B b
(n 1)

6.59 1.35
(n 3)

6.59 1.35
(n 3)

9.11 1.12 D
(n 4)

10.13 0.39 b
(n 2)

8.09 0.32 C a
(n 2)

10.01 0.86 b
(n 1)
6.72 0.47 c
(n 2)
8.14 1.39 C
(n 7)

6.58 0.85
(n 2)

6.58 0.85
(n 2)

8.47 1.01 a
(n 2)

8.29 1.22
(n 2)

No. of antibiotics to which Salmonella strains are resistant:

10.13 1.02
(n 4)

10.13 1.02
(n 4)

10.73 0.32
(n 1)

10.73 0.32
(n 1)

6.70 1.60 a
(n 36)
8.96 1.58 bc
(n 3)
10.13 0.39 b
(n 2)
5.55 0.14 a
(n 6)
6.74 0.14 a
(n 5)
8.47 1.01 c
(n 2)
6.15 0.13 a
(n 3)
10.01 0.86 b
(n 1)
6.72 0.47 a
(n 2)
6.90 1.65
(n 60)

Avg

Means in the same row without capital letters in common are significantly (P 0.05) different. Means in the same column without lowercase letters in common are significantly (P 0.05)
different.
b No. of strains; each strain was tested three times in separate days.
c , no data.

5.59 0.15
(n 3)

6.15 0.13 d
(n 3)

5.51 0.11 A a
(n 3)
6.74 0.14 c
(n 5)

5.75 0.36 A aa
(n 21)b
7.33 0.49 A b
(n 1)

Paratyphi B

Derby

Agona

Virchow

Poona

Infantis

Newport

Typhimurium

Enteritidis

Serotypes of
Salmonella

TABLE 2. D-values (s) for Salmonella in peptone water with 1,000 ppm acidified sodium chlorite, with respect to serotype and antibiotic resistance pattern

J. Food Prot., Vol. 70, No. 8

VARIATION OF SALMONELLA STRAINS IN RESISTANCE TO DECONTAMINANTS

1837

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CAPITA

J. Food Prot., Vol. 70, No. 8

FIGURE 1. First-order inactivation kinetics of Salmonella in peptone water in the presence of 1,000 ppm of acidified sodium chlorite.
(A) Salmonella enterica. (B) Salmonella Enteritidis. (C) Salmonella Typhimurium. (D) Salmonella Infantis. Black rhombus, sensitive
strains; asterisk, resistant strains; white square, multiresistant strains.

tions. After dilution, 1-ml samples were taken and pour plated
with tryptic soy agaryeast extract. Two pour plates per dilution
were prepared, inverted, and incubated at 37C for 48 h. Each
experiment was repeated three times on separate days.
Statistical analysis. The average number of colonies from
the duplicate plates was recorded for each sample. The survivor
curve was represented as log(N/N0) kt, where N is the number
of surviving Salmonella after treatment time t (s), N0 is the number of microorganisms at time t 0, and k is the inactivation rate
constant (s1). The k values for individual survivor curves were
obtained from the lineal regression of log(N/N0) versus time as
negative slope. The 95% confidence intervals were calculated for
each data point. To compare the resistance of strains, the decimal
reduction time (D-value, time in seconds required to destroy the
microbial population in a sample by 90% at the described compound concentration) was calculated as D 2.303/k (20). D-values were compared for statistical significance with the MannWhitney U test. Pearsons correlation coefficient was used to determine the relationship between antibiotic resistance (number of
antibiotics to which the strains are resistant) and D-values. Data
were analyzed using the Statistica for Windows, release 6.0 (Statsoft, Inc., Tulsa, Okla.), software package.

RESULTS
The chemical inactivation process followed first-order
kinetics for the range studied in this investigation (r2
0.96). Therefore, decimal reduction times (D-values, seconds) were calculated from the survival curve slopes. Observed 95% confidence limits varied from mean 0.03 to
mean 0.65 log(N/N0) for data points in the graphs.

The D-values in presence of 1,000 ppm ASC determined for each serotype and number of antibiotics to which
strains are resistant are shown in Table 2. Resistance of
Salmonella strains to ASC varied among serotypes, with
Typhimurium, Newport, and Derby showing the highest Dvalues. The lowest ASC D-values were observed for Salmonella serotypes Enteritidis, Infantis, and Paratyphi B.
Variations in resistance to ASC among phage types (determined in sensitive Salmonella Enteritidis strains) were
small, with D-values ranging from 5.75 0.55 s (phage
type [PT] 1) to 5.99 0.25 s (PT 4b) (P 0.05).
There were wide variations in the resistance to ASCrelated to antibiotic resistance profiles, with average D-values ranging from 5.59 0.15 s (strains resistant to one
antibiotic) to 10.73 0.32 s (strain resistant to nine antibiotics). Figure 1 shows survival curves of antibiotic-sensitive, -resistant (one antibiotic) and multiresistant (two or
more antibiotics) strains in presence of ASC. Average Dvalues for multiresistant Salmonella strains (8.37 1.69 s)
were higher (P 0.05) than those of resistant (5.59 0.15
s) and sensitive (5.96 0.54 s) strains. Similar findings
were obtained with Salmonella Enteritidis (8.04 1.70 s
for multiresistant versus 5.75 0.36 s for sensitive, P
0.05) and Salmonella Typhimurium (9.77 1.24 s for multiresistant as compared with 7.33 0.49 s for sensitive, P
0.05) isolates. No differences were observed among Salmonella Infantis (D-values of 5.51 0.11 s and 5.59
0.15 s for sensitive and resistant isolates, respectively).

VARIATION OF SALMONELLA STRAINS IN RESISTANCE TO DECONTAMINANTS

For interpretation, see Table 2.

12.64 0.26
A

12.44 0.91

Enteritidis
Typhimurium
Newport
Infantis
Poona
Virchow
Agona
Derby
Paratyphi B
Salmonella
(all strains)

12.82 0.73 A a

12.61 0.12 A a

11.99 0.42 A a 12.64 0.26

12.19 0.47 a

10.66 0.57 b

13.14 0.61

13.32 0.74

13.47 0.52

13.33 0.86

12.29 0.66

13.51 0.79

13.45 0.50

12.79 0.92

0.80
0.52
0.19
0.48
0.47
0.76
0.57
0.72
0.87

13.33 0.63
12.78 0.46

a 13.32 0.74
a

13.52 0.75 A a 13.57 0.88 A a 12.29 0.66

13.43 0.19 a

13.45 0.76 a

13.80 0.72 a

12.72 0.87 a

a 13.51 0.79

13.45 0.50

13.02
12.95
13.43
12.31
12.19
13.45
10.66
13.80
12.72

Avg
9
7
6
5
4

No. of antibiotics to which Salmonella strains are resistant:

3
2
1
0
Serotypes of
Salmonella

TABLE 3. D-value (s) for Salmonella in peptone water with 1.2% trisodium phosphate, with respect to serotype and antibiotic resistance patterna

1839

Correlation coefficient indicated a highly significant

positive relationship between D-values and the number of
antibiotics to which strains are resistant in the case of S.
enterica (considering all 60 strains simultaneously; 0.775,
P 0.001), Salmonella Enteritidis (0.820, P 0.001), and
Salmonella Typhimurium (0.906, P 0.001).
The D-values calculated for Salmonella in the presence
of TSP and CA are presented in Tables 3 and 4, respectively. TSP resistance varied only minimally according to
serotype, with Salmonella Agona showing the lowest Dvalues. As indicated for ASC, minimal variations among
phage types were observed in resistance to TSP, with Dvalues ranging from 12.33 0.87 s (PT 1) to 13.07 0.36
s (PT 4) (P 0.05). Variation in TSP resistance among
antibiotic resistance patterns was also scarce, with only significant differences in D-values among Salmonella Infantis
strains (higher in antibiotic-resistant than in -sensitive
strains). However, when grouping antibiotic-sensitive, -resistant, and multiresistant strains (Fig. 2), variations among
strains according to TSP resistance were found, with higher
(P 0.05) average D-values observed for multiresistant
(13.28 0.77 s) than for resistant (12.63 0.26 s) or
sensitive (12.44 0.91 s) Salmonella strains. When each
Salmonella serotype was considered separately, resistance
variations were also related to antibiotic resistance patterns.
Thus, D-values observed were 13.29 0.82 s (multiresistant) versus 12.82 0.73 s (sensitive) (P 0.05) for Salmonella Enteritidis, and 13.12 0.57 s (multiresistant) as
compared with 12.61 0.12 s (sensitive) (P 0.05) for
Salmonella Typhimurium. No differences were observed
between resistant (12.64 0.26 s) and sensitive (11.99
0.42 s) Salmonella Infantis strains. Pearsons correlation coefficients between TSP D-values and the number of antibiotics to which strains were resistant showed high values
only for Salmonella Typhimurium (0.741, P 0.05) and
Salmonella Infantis (0.700, P 0.05) strains.
Neither serotype nor antibiotic resistance influenced resistance of Salmonella to CA treatment, with similar Dvalues observed for all trials (Table 4; Figure 3). Differences in resistance to CA among phage types varied only
minimally, with values ranging from 11.90 0.93 s (PT
1) to 12.44 0.23 s (PT 4b) (P 0.05). No significant
Pearsons correlation coefficients were obtained, either for
Salmonella or individual serotypes, between D-values in
the presence of CA and the number of antibiotics to which
strains were resistant. Pearsons correlation coefficient likewise revealed no relationship between the resistance levels
of Salmonella to the three compounds tested.

a
ab
a
b
b
a
c
a
ab

J. Food Prot., Vol. 70, No. 8

DISCUSSION
Although previous research has tested the resistance of
Salmonella to ASC, TSP, and CA (8, 9), no studies have
been carried out to investigate the intraspecific variation of
the resistance to decontaminant compounds. The main purpose of this study was a preliminary screening to detect
differences between the resistance levels of Salmonella
strains to chemical decontaminants for poultry and to relate
these to serotype, phage type, and antibiotic resistance patterns. It should be pointed out that this research was per-

1840

J. Food Prot., Vol. 70, No. 8

12.24 0.47
A
a

For interpretation, see Table 2.

12.17 0.57
A

12.12 0.83

Enteritidis
Typhimurium
Newport
Infantis
Poona
Virchow
Agona
Derby
Paratyphi B
Salmonella
(all strains)

11.97 0.90 A a

12.72 0.69 A a

12.26 0.71 A a 12.17 0.57

12.56 0.51 a

12.08 0.54 a

12.47 0.31

12.02 1.04

12.60 1.04

12.19 0.94

12.14 0.92

12.51 0.23

12.20 0.81

0.83
0.49
0.59
0.63
0.51
1.51
0.54
0.49
0.82

12.51 0.23

12.14 0.92

12.08 0.87 A a 12.19 0.94

12.97 1.51 a

12.70 0.49 a

12.72 0.82 a

12.12 0.38 A a 12.47 0.31

12.48 0.62 A a

12.11 0.57 A a

11.94 0.59 a

6
5
4

No. of antibiotics to which Salmonella strains are resistant:

3
2
1
0
Serotypes of
Salmonella

TABLE 4. D-value (s) for Salmonella in peptone water with 25% citric acid, with respect to serotype and antibiotic resistance patterna

12.06
12.57
11.94
12.21
12.56
12.97
12.08
12.70
12.72

Avg

a
a
a
a
a
a
a
a
a

CAPITA

formed to compare rates of bacterial inactivation for different Salmonella strains, but not intended to simulate natural environments or to compare chemical compounds.
Thus, chemical concentrations employed here may not be
directly comparable with those reported elsewhere.
The first-order kinetics found in the study being presented here for inactivation of Salmonella strains by ASC,
TSP, and CA is a result congruent with findings of most
authors testing the antimicrobial effect of chemical biocides
(13, 14).
Our results suggest that the resistance of Salmonella to
ASC is substantially different among serotypes and antibiotic resistance patterns. The high level of resistance to
ASC found in the present study among Salmonella Typhimurium strains is of great interest from the standpoint of
public health because this serotype is among the most dangerous for humans according to Sarwari et al. (29). These
authors developed a mathematical model to predict ability
to cause human illness, finding that Salmonella Typhimurium had the highest score of the seven serotypes they
compared. It is not known, however, if the Salmonella Typhimurium strains tested in the present research are atypical
clones with extreme resistance to ASC, or if their high Dvalues are a general phenomenon, because only three isolates from this serotype were available for examination during this study.
The antimicrobial action of ASC is attributed to chlorous acid, which is derived from the conversion of chlorite
ion into acid form under acidic conditions. Chlorous acid
kills microorganisms by direct action on the cellular membrane and by oxidation of cellular constituents (5, 30). The
relationship between ASC and antibiotic resistance of Salmonella has not been tested so far. However, cross-resistance (mediated by a genetic linkage) to antibiotics and to
chlorine biocides other than ASC has been previously reported (19, 28). Similarities in the actions of chlorine biocides and those of antibiotics include damaging of membranes (chlorhexidine salts, triclosan, polymyxins, streptomycin), or other cytological effects (chloroacetamide, lactams, fluoroquinolones, novobiocin) (22, 24, 27).
The antimicrobial effect of TSP results from a combination of several factors. First, there is the high pH (12.0
to 13.0) of TSP solutions, which appears to disrupt fatty
molecules in the cell membrane, causing the bacterial cells
to leak intracellular fluid. Second, there is their ionic
strength, which can cause bacterial cell autolysis. The ability to remove fat films and to have a surfactant or detergent
effect also contributes to the decontaminant effect of TSP
on red meat and poultry carcasses (4, 8, 25). Investigations
into the relationship between resistance to TSP and the serotype, phage type, or antibiotic resistance of Salmonella
have not been carried out so far. The results being presented
in the present study suggest a minimal (although significant) intraspecific variation in resistance to TSP of this bacterium, relating principally to the pattern of resistance to
antibiotics.
The bactericidal effect of CA and other organic acids
is largely due to the ability of the undissociated form of
these compounds to diffuse through the cell membrane into

J. Food Prot., Vol. 70, No. 8

VARIATION OF SALMONELLA STRAINS IN RESISTANCE TO DECONTAMINANTS

1841

FIGURE 2. First-order inactivation kinetics of Salmonella in peptone water in the presence of 1.2% trisodium phosphate. For interpretation, see Figure 1.

FIGURE 3. First-order inactivation kinetics of Salmonella in peptone water in the presence of 25% citric acid. For interpretation, see
Figure 1.

1842

CAPITA

the cytoplasm. Since the intracellular pH is usually higher

than that of the external environment, some of the acid
dissociates, tending to cause accumulation of protons and
anions in the cell cytoplasm. This acidification is the primary cause of the antimicrobial activity of organic acids,
while some effects of the anions has been also suggested
as a cause (8, 32). In the present study, no variation in the
resistance of Salmonella to CA was observed among
strains. These results are congruent with observations by
other authors (2, 11, 33), who reported no difference in acid
resistance between antibiotic-resistant and -susceptible Salmonella strains. On the other hand, the lack of variation in
acid resistance among serotypes observed in the present
study does not agree with findings by other authors (17,
21), who observed that Salmonella Typhimurium is able to
survive a wider range of acid stress than are other Salmonella serotypes.
The absence of any relationship (no significant Pearsons correlation coefficient) among the levels of resistance
by Salmonella to the three compounds tested is a result that
was to be expected because the mechanisms of inactivation
are intrinsically different, as indicated in previous paragraphs.
Results from the present study suggest that when testing chemical decontamination procedures (especially ASC
treatments), validation trials should be conducted using
control Salmonella strains which are recognized as having
a resistance to the chemicals used, which is at the higher
end of the spectrum for the pathogenic species, in order to
ensure that appropriate concentrations are used to inactivate
all strains of the target pathogens. However, further research
on a larger numbers of antibiotic-sensitive, -resistant, and
multiresistant strains from various different serotypes and
phage types is required to confirm these findings.

J. Food Prot., Vol. 70, No. 8

7.

8.

9.

10.

11.

12.

13.
14.

15.

16.

17.

ACKNOWLEDGMENTS
The author thanks the Spanish Ministerio de Sanidad y Consumo
(Instituto de Salud Carlos III, Project FIS PI 040722) and the Junta de
Castilla y Leon (Project SAN/1052/LE02/05) for their financial support.

REFERENCES
1.

2.

3.

4.

5.

6.

Anonymous. 30 April 2004. Regulation (EC) no. 853/2004 of the

European Parliament and of the Council of 29 April 2004, laying
down specific hygiene rules for the hygiene of foodstuffs. Off. J.
Eur. Communities L 139 47:55. Available at: http://europa.eu.int/
eur-lex/en/archive/2004/l13920040430en.html. Accessed 9 February 2007.
Bacon, R. T., J. N. Sofos, P. A. Kendall, K. E. Belk, and G. C. Smith.
2003. Comparative analysis of acid resistance between susceptible
and multi-antimicrobial-resistant Salmonella strains cultured under
stationary-phase acid tolerance-inducing and noninducing conditions. J. Food Prot. 66:732740.
Braoudaki, M., and A. C. Hilton. 2004. Adaptative resistance to biocides in Salmonella enterica and Escherichia coli O157 and crossresistance to antimicrobial agents. J. Clin. Microbiol. 42:7378.
Capita, R., C. Alonso-Calleja, M. C. Garca-Fernandez, and B. Moreno. 2002. Review: Trisodium phosphate treatment for decontamination of poultry. Food Sci. Tech. Int. 8:1124.
Castillo, A., L. M. Lucia, G. K. Kemp, and G. R. Acuff. 1999.
Reduction of Escherichia coli O157:H7 and Salmonella typhimurium
on beef carcass surfaces using acidified sodium chloride. J. Food
Prot. 62:580584.
Clinical and Laboratory Standards Institute (CLSI). 2002. Perfor-

18.

19.

20.
21.

22.
23.
24.

25.

26.

27.

mance standards for antimicrobial disk and dilution susceptibility test

for bacteria isolated from animals. Approved standard M31-A2.
NCCLS, Wayne, Pa.
Del Ro, E., R. Capita, M. Prieto, and C. Alonso-Calleja. 2006. Comparison of pathogenic and spoilage bacterial levels on refrigerated
poultry parts following treatment with trisodium phosphate. Food
Microbiol. 23:195198.
Del Ro, E., R. Muriente, M. Prieto, C. Alonso-Calleja, and R. Capita. 2007. Effectiveness of trisodium phosphate, acidified sodium
chlorite, citric acid, and peroxyacids against pathogenic bacteria on
poultry during refrigerated storage. J. Food Prot., in press.
Del Ro, E., M. Panizo-Moran, M. Prieto, C. Alonso-Calleja, and R.
Capita. 2007. Effect of various chemical decontamination treatments
on natural microflora and sensory characteristics of poultry. Int. J.
Food Microbiol. 115:268280.
Dincer, A. H., and T. Baysal. 2004. Decontamination techniques of
pathogen bacteria in meat and poultry. Crit. Rev. Microbiol. 30:197
2004.
Duffy, G., C. Walsh, I. S. Blair, and D. A. McDowell. 2006. Survival
of antibiotic-resistant and antibiotic-sensitive strains of E. coli O157
and E. coli O26 in food matrices. Int. J. Food Microbiol. 109:179
186.
Echeita, A., A. Aladuena, R. Gonzalez-Sanz, R. Dez, M. de la Fuente, F. Cerdan, M. Arrollo, and R. Gutierrez. 2005. Analisis de las
cepas de Salmonella spp. aisladas de muestras clnicas de origen
humano en Espaa. Anos 2002 y 2003 (I). BES 13:7376.
Erkmen, O. 2004. Hypochlorite inactivation kinetics of Listeria monocytogenes in phosphate buffer. Microbiol. Res. 159:167171.
Erkmen, O., and H. Karaman. 2001. Kinetic studies on the high
pressure carbon dioxide inactivation of Salmonella typhimurium. J.
Food Engin. 50:2528.
European Food Safety Authority. 2005. Opinion of the scientific panel on food additives, flavourings, processing aids and materials in
contact with food (AFC) on a request from the Commission related
to treatment of poultry carcasses with chlorine dioxide, acidified sodium chlorite, trisodium phosphate and peroxyacids. EFSA J. 297:
127.
Federation of Veterinarians of Europe. 2005. Public health. Implementing measures of the hygiene package, p. 46. May newsletter.
Federation of Veterinarians of Europe, Brussels.
Foster, J. W. 1995. Low pH adaptation and the acid tolerance response of Salmonella typhimurium. Crit. Rev. Microbiol. 21:215
237.
Hernandez-Pezzi, G., A. Torres, P. Ordonez, and C. Cevallos. 2004.
Brotes de enfermedades treansmitidas por alimentos. Espana, 1993
2002 (excluye brotes hdricos). BES 12:289296.
Langsrud, S., M. S. Sidhu, E. Heir, and A. L. Holck. 2003. Bacterial
disinfectant resistancea challenge for the food industry. Int. Biodet. Biodegr. 51:283290.
Lewis, J. C. 1956. The estimation of decimal reduction times. Appl.
Environ. Microbiol. 4:211221.
Leyer, G. J., and E. A. Johnson. 1993. Acid adaptation promotes
survival of Salmonella spp. in cheese. Appl. Environ. Microbiol. 58:
20752080.
Meyer, B. 2006. Does microbial resistance to biocides create a hazard to food hygiene? Int. J. Food Microbiol. 112:275279.
NCCLS. 2000. Performance standards for antimicrobial disk susceptibility tests. Approved standard M2-A7. NCCLS, Wayne, Pa.
Oren Gradel, K., L. Randall, A. R. Sayers, and R. H. Davies. 2005.
Possible association between Salmonella persistence in poultry houses and resistance to commonly used disinfectants and a putative role
of mar. Vet. Microbiol. 107:127138.
Oyarzabal, O. A. 2005. Reduction of Campylobacter spp. by commercial antimicrobials applied during the processing of broiler chickens: a review from the United States perspective. J. Food Prot. 68:
17521760.
Padungtod, P., and J. B. Kaneene. 2006. Salmonella in food animals
and humans in northern Thailand. Int. J. Food Microbiol. 108:346
354.
Potenski, C. J., M. Gandhi, and K. R. Matthews. 2003. Exposure of

J. Food Prot., Vol. 70, No. 8

VARIATION OF SALMONELLA STRAINS IN RESISTANCE TO DECONTAMINANTS

Salmonella Enteritidis to chlorine or food preservatives increases

susceptibility to antibiotics. FEMS Microbiol. Lett. 220:181186.
28. Russell, A. D. 2003. Biocide use and antibiotic resistance: the relevance of laboratory findings to clinical and environmental situations. Lancet Infect. Dis. 3:794803.
29. Sarwari, A., S. Magder, P. Levine, A. McNamara, S. Knower, G.
Armstrong, R. Etzel, J. Hollingsworth, and J. Morris. 2001. Serovar
distribution of Salmonella isolates found in humans. J. Infect. Dis.
183:1295.
30. Scientific Committee on Veterinary Measures relating to Public
Health. 2003. Opinion of the Scientific Committee on Veterinary
Measures relating to public health on the evaluation of antimicrobial

1843

treatments for poultry carcasses, April 2003. European Commission,

Brussels.
31. Tavechio, A. T., A. C. R. Ghilardi, J. T. M Peresi, T. O. Fuzihara,
E. K. Yonamine, M. Jakabi, and S. A. Fernandez. 2002. Salmonella
serotypes isolated from nonhuman sources in Sao Paulo, Brazil, from
1996 through 2000. J. Food Prot. 65:10411044.
lvarez, S. Condon, and J. Raso. 2005. Inac32. Virto, R., D. Sanz, I. A
tivation kinetics of Yersinia enterocolitica by citric and lactic acid
at different temperatures. Int. J. Food Microbiol. 103:251257.
33. Walsh, C., D. Duffy, J. J. Sheridan, S. Fanning, I. S. Blair, and D.
A. McDowell. 2005. Thermal resistance of antibiotic-resistant and
antibiotic-sensitive Salmonella spp. on chicken meat. J. Food Saf.
25:288302.