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Applied and Environmental Microbiology, December 2000, p. 5248-5252, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
PCR Detection of Salmonella enterica
Serotype Montevideo in and on Raw Tomatoes Using Primers Derived
from hilA
Xuan
Guo,1
Jinru
Chen,1
Larry R.
Beuchat,1,* and
Robert E.
Brackett1,2
Center for Food Safety and Quality
Enhancement, Department of Food Science and Technology, The University
of Georgia, Griffin, Georgia 30223-1797,1 and
Office of Plant, Dairy Foods and Beverages, U.S. Food and
Drug Administration, Washington, D.C. 202042
Received 26 June 2000/Accepted 14 September 2000
 |
ABSTRACT |
Salmonellae have been some of the most frequently reported
etiological agents in fresh-produce-associated outbreaks of human infections in recent years. PCR assays using four innovative pairs of
primers derived from hilA and sirA, positive
regulators of Salmonella invasive genes, were developed to
identify Salmonella enterica serotype Montevideo on and in
tomatoes. Based on examination of 83 Salmonella strains and
22 non-Salmonella strains, we concluded that a pair of
hilA primers detects Salmonella specifically.
The detection limits of the PCR assay were 101 and
100 CFU/ml after enrichment at 37°C for 6 and 9 h,
respectively. When the assay was validated by detecting S. enterica serotype Montevideo in and on artificially inoculated
tomatoes, 102 and 101 CFU/g were detected,
respectively, after enrichment for 6 h at 37°C. Our results
suggest that the hilA-based PCR assay is sensitive and
specific, and can be used for rapid detection of Salmonellae in or on
fresh produce.
| |
INTRODUCTION |
Salmonellae are some of the most
prevalent food-borne pathogens in United States. They are estimated to
cause approximately 1.5 million cases of infection, 15,000 hospitalizations, and 500 deaths annually (25).
Historically, salmonellosis has most often been associated with
consumption of contaminated foods of animal origin, such as poultry,
eggs, meat, and dairy products. Changes in agronomic practices and
dietary habits and increased importation of fresh produce are thought
to contribute to the increased numbers of outbreaks associated with
fruits and vegetables in recent years (3). Outbreaks of
salmonellosis have been linked to tomatoes (12; R. C. Wood, C. Hedberg, and K. White, Abstr. Epidemic Intelligence Service 40th Annu.
Conf., p. 69, 1991), seed sprouts (23, 28; C. A. Van Beneden,
W. E. Keene, D. H. Werker, A. S. King, P. R. Cieslak, K. Hedberg, R. A. Strang, A. Bell, and D. Fleming, Abstr. 36th Intersci. Conf. Antimicrob. Agents Chemother., abstr. K47, p. 259, 1996), watermelons (8, 10, 16), cantaloupes (11; A. A. Ries, S. Zaza, C. Langkop, R. V. Tauxe, and P. A. Blake, Program Abstr. 30th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 915, p. 238, 1990), orange juice (K. Cook, D. Swerdlow, T. Dobbs, J. Wells, N. Puhr, G. Hlady, C. Genese, L. Finelli, B. Toth, D. Bodager, K. Pilot, and P. Griffin, Abstr. 36th Intersci. Conf.
Antimicrob. Agents Chemother., abstr. K49, p. 259, 1996), and apple
cider (9).
Consumption of fresh tomatoes was epidemiologically linked to 176 cases
of Salmonella javiana infection in Illinois, Michigan, Minnesota, and Wisconsin in 1990 (Wood et al., Abstr. Epidemic Intelligence Service 40th Annu. Conf.). In 1993, tomatoes were identified as the vehicle for a multistate outbreak of Salmonella enterica serotype Montevideo infection (12). Zhuang et
al. (32) described conditions that influence survival and
growth of S. enterica serotype Montevideo on the surfaces of
intact tomatoes. Rapid growth occurred in chopped ripe tomatoes (pH
4.1 ± 0.1) at the ambient temperature. S. enterica
serotype Enteritidis, S. enterica serotype Infantis, and
S. enterica serotype Typhimurium have been reported to grow
in fresh cut tomatoes (pH 3.99 to 4.37) at 22 and 30°C
(4).
Contamination of fresh produce with salmonellae may occur at any point
along the farm-to-table continuum, and salmonellae probably occur
intermittently at low levels together with the diverse natural flora.
Thus, rapid and sensitive methods for detecting salmonellae are in
great demand in order to assure produce safety. One of the most
promising methods for detecting salmonellae is based on the PCR, which
combines simplicity with specificity and sensitivity for detecting the
pathogens in food. Several PCR assays have been developed by targeting
various Salmonella genes, such as invA (29,
31), 16S rRNA (19), agfA (14),
and viaB (18), and virulence-associated plasmids
(24, 30). These PCR assays are used mainly for detecting
salmonellae in poultry, meat, and milk samples (5, 7, 13,
22). Few of the assays have been used to detect the pathogens in
fresh produce.
Invasion is an important factor influencing the virulence of
Salmonella spp. The invasive phenotype is determined by a
large cluster of genes in Salmonella pathogenicity island 1 (SPI1) (26), which is present in all invasive strains of
Salmonella (15). One of the SPI1 virulence genes,
hilA, is a positive transcriptional regulator of several
invasion genes (6). sirA, however, is a gene that
is located outside SPI1 (21) and is known as a global regulator of invasion genes as well as several other genes that mediate
enteropathogenesis (2).
In this study, PCR assays were developed by using primers derived from
hilA and sirA. The specificities and
sensitivities of the assays were evaluated by using pure cultures and
were validated by detecting salmonellae in and on artificially
contaminated tomatoes.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
Eighty-three
Salmonella strains and 22 non-Salmonella strains
(Table 1) used in this study were
obtained from the laboratory collections of Jinru Chen and Larry
Beuchat at the Center for Food Safety and Quality Enhancement,
University of Georgia. Before they were subjected to PCR, strains were
retrieved from frozen stock cultures and grown in brain heart infusion
(BHI) broth (Difco Laboratories, Detroit, Mich.) at 37°C for 16 to
18 h. Stock cultures were maintained on BHI agar at 4°C
throughout the study.
DNA preparation.
Both crude DNA and purified DNA were used
in the PCR assays. Crude DNA was prepared by boiling bacterial cell
suspensions. One milliliter of an overnight bacterial culture was
centrifuged at 12,000 × g for 2 min (model 5415C
microcentrifuge; Eppendorf, Hamburg, Germany). Pellets were resuspended
in 200 µl of sterile distilled water, boiled for 10 min, and
centrifuged as described above. A 5-µl aliquot was used as a template
for PCR. When purified DNA was prepared, a DNeasy tissue kit (Qiagen,
Valencia, Calif.) was used according to the manufacturer's instructions.
Specificity of PCR assays for Salmonella
strains.
Four oligonucleotide primers (Table
2) derived from hilA (GenBank
accession no. U25352) and sirA (GenBank accession no. U67869) were designed in this study and were synthesized by GIBCO BRL
(Rockville, Md.). The specificities of all primers were tested by using
83 Salmonella strains and 22 non-Salmonella
strains.
Each 50-µl PCR mixture contained PCR buffer, deoxynucleoside
triphosphates (0.05 mM each), primers (0.25 µM each),
Taq
polymerase
(1 U; Roche Diagnostics, Indianapolis, Ind.), and DNA
template
(5 µl, equivalent to approximately 10
7 CFU/ml).
PCR were performed with DNA thermal cycler (model 480;
Perkin-Elmer, Norwalk, Conn.) by using one cycle at 94°C for 5
min,
followed by 30 cycles of 94°C for 2 min, 62°C for 1 min,
and
72°C for 1 min and final extension at 72°C for 10 min. The
PCR
amplicons were analyzed by gel electrophoresis on a 1% agarose
(GIBCO BRL) gel in 1× TBE buffer (0.089 M Tris-borate, 0.002 M
EDTA; pH 8.0). The gel was stained with ethidium bromide and visualized
with the Gel Doc System 2000 (Bio-Rad Laboratories, Hercules,
Calif.).
Limits of detection of HILA2-based PCR.
S.
enterica serotype Montevideo G4639, isolated from a patient
infected in the 1993 tomato outbreak (32), was used for the sensitivity study and for artificial inoculation of tomatoes. The
organism was grown at 37°C for 16 to 18 h in BHI broth with shaking in an incubator shaker (model G-24; New Brunswick Scientific, Edison, N.J.). Cells were harvested when the culture optical density at
600 nm was approximately 1.0 (Novaspec II spectrophotometer; Pharmacia
Biotech, Cambridge, United Kingdom), plated on BHI agar, and incubated
at 37°C for 24 h before colonies were counted. The cultures were
serially diluted to give suspensions containing 100 to
109 CFU/ml in 10-fold increments. Crude DNA was prepared
and amplified by PCR. Purified DNA from the S. enterica
serotype Montevideo culture was diluted to the equivalent of
100 to 109 CFU/ml for PCR amplification.
The effect of enrichment time on sensitivity was investigated.
Salmonella cultures (10
9 CFU/ml) were serially
diluted to give 10
0 to 10
8 CFU/ml. One-hundred
microliters was transferred to 900 µl of
BHI broth, which was then
incubated at 37°C with shaking for 3,
6, and 9 h. The cell
cultures were harvested and the DNA were
amplified by PCR by using the
procedures described
above.
PCR detection with artificially contaminated tomatoes.
Raw,
ripe Roma tomatoes whose average weight was 75 g were purchased
from a local grocery store and divided into two groups for surface or
internal inoculation. S. enterica serotype Montevideo was
grown in BHI broth at 37°C for 16 to 18 h with shaking and was
harvested when the optical density at 600 nm reached 1.0, which
corresponded to 109 CFU/ml. The culture was serially
diluted in sterile 0.1% peptone water, and 50 µl was used for
inoculation. Negative controls consisted of sterile distilled water
instead of a cell suspension.
For surface contamination, each tomato was inoculated with 50 µl of
an
S. enterica serotype Montevideo suspension. The sizes
of
the inocula ranged from 10
0 to 10
5 CFU per
tomato. Each inoculum was deposited in small drops to
facilitate
distribution and rapid drying. Inoculated tomatoes
were then placed in
a laminar flow hood and air dried for 2 h
at 22 ± 1°C.
Each tomato was placed in a stomacher bag with 20
ml of sterile 0.1%
peptone water and massaged by hand for 2 min.
The rinse water was
transferred into 50-ml Falcon centrifuge tubes
and centrifuged at
12,000 ×
g for 10 min (model 5810R centrifuge;
Eppendorf). The pellets were resuspended in sterile 0.1% peptone
water. Five milliliters of suspension was combined with 5 ml of
BHI
broth for enrichment. Cells were harvested after 6 h of
enrichment,
and a PCR was performed. Plate counts were obtained by
using BHI
agar and bismuth sulfite agar (Difco) to estimate the numbers
of
cells.
For internal inoculation,
S. enterica serotype Montevideo
was injected into tomatoes around the stem scar areas by using a
20-µl pipette tip; the sizes of the inocula were ca. 10
0,
10
1, 10
2, 10
3, 10
4, and
10
5 CFU/tomato. Each inoculated tomato was placed into a
stomacher
filter bag with 50 ml of sterile 0.1% peptone water and
stomached
for 1 min at normal speed by using a Stomacher 400 (Seward,
London,
United Kingdom). The liquid portion was transferred into a
50-ml
Falcon centrifuge tube and centrifuged at 3,000 ×
g for 5 min;
the supernatant fluid was centrifuged at
3,000 ×
g for 5 min before
it was transferred to a
second tube and centrifuged at 10,000
×
g for 10 min.
Plate counting and PCR were done as described
above.
| |
RESULTS |
Specificity of the PCR assay for bacterial cultures.
Four
pairs of primers (HILA1, HILA2, SIRA1, and SIRA2) were evaluated, and
one of them, HILA2, detected only Salmonella. All 83 Salmonella strains tested positive, whereas
non-Salmonella strains were not detected. Figure
1 shows representative PCR products from
Salmonella and non-Salmonella strains obtained
with HILA2. The other three pairs of primers, HILA1, SIRA1, and
SIRA2, generated nonspecific bands (data not shown). HILA1 yielded
nonspecific bands with Yersinia enterocolitica, whereas
SIRA1 produced nonspecific products from Escherichia coli,
Shigella sonnei, Y. enterocolitica, and
Pseudomonas fluorescens. The false-positive results obtained with SIRA2 were associated with E. coli, Shigella
sonnei, Klebsiella pneumoniae, Serratia
marcescens, and Y. enterocolitica.
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FIG. 1.
Specificity of HILA2-based PCR assay for
Salmonella: gel electrophoresis of PCR products in 1%
agarose in 1× TBE buffer. (A) Salmonella strains. Lanes 1 and 22, 100-bp DNA ladders (GIBCO BRL); lanes 2 through 20, PCR
products amplified from S. enterica serotype Anatum,
S. enterica serotype Baildon, S. enterica
serotype Cubana, S. enterica serotype Enteritidis, S. enterica serotype Gaminara, S. enterica serotype
Hartford, S. enterica serotype Heidelberg, S. enterica serotype Infantis, S. enterica serotype
Michigan, S. enterica serotype Montevideo, S. enterica serotype Muenchen, S. enterica serotype
Newport, S. enterica serotype Oranienburg, S. enterica serotype Panama, S. enterica serotype Poona,
S. enterica serotype Saintpaul, S. enterica
serotype Thompson, S. enterica serotype Typhimurium, and
S. enterica serotype Typhimurium DT 104, respectively; Lane
21, negative control. (B) Non-Salmonella strains. Lanes 1 and 22, 100-bp DNA ladders (GIBCO BRL); lane 2, S. enterica
serotype Typhimurium; lanes 3 through 20, the negative results obtained
with Aeromonas sobria (lane 3), Escherichia coli ATCC 10789 (lane 4), Escherichia coli O157:H7 (lanes 5 and 6),
Enterobacter aerogenes (lane 7), Klebsiella
pneumoniae (lane 8), Serratia marcescens (lane 9),
Shigella dysenteriae non-type I (lane 10), Shigella
sonnei, (lanes 11 through 13), Staphylococcus aureus,
(lanes 14 and 15), Proteus vulgaris (lane 16),
Pseudomonas fluorescens (lane 17), Yersinia
enterocolitica (lanes 18 through 20); lane 21, negative control.
|
|
Limits of detection of PCR assay for Salmonella
cultures.
The sensitivity of the HILA2-based PCR assay was
investigated. Figure 2A shows that crude
DNA from a suspension containing 105 CFU/ml yielded
positive amplification, whereas pure DNA extracted with the Qiagen kit
provided better sensitivity, with a detection limit of 104
CFU/ml (Fig. 2B). The detection limits were as low as 101
and 100 CFU/ml with 6- and 9-h enrichments, respectively
(Fig. 3).
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FIG. 2.
Limits of detection of HILA2-based PCR assay. (A) PCR
products amplified from crude DNA. Lane 1, 100-bp DNA ladders; lanes 2 through 10, 108, 107, 106,
105, 104, 103, 102,
101, and 100 CFU/ml, respectively; lane 11, negative control. (B) PCR products amplified from purified DNA. Lane 1, 100-bp DNA ladders; lanes 2 through 10, DNA equivalent to
108, 107, 106, 105,
104, 103, 102, 101, and
100 CFU/ml, respectively; lane 11, negative control.
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FIG. 3.
Limits of detection of HILA2-based PCR assay with
different enrichment times. Cell cultures containing 109
CFU/ml were serially diluted to concentrations of 108 to
100 CFU/ml, and 100-µl portions of the dilutions were
transferred to 900-µl portions of BHI broth, which were then
incubated at 37°C with shaking for 3, 6 and 9 h. The cells were
treated, and DNA was amplified by PCR. Lanes 1 and 21, 100-bp DNA
ladders; lanes 2 through 7, PCR products from preparations containing
105, 104, 103, 102,
101, and 100 CFU/ml, respectively, after 3 h of enrichment; lanes 8 through 13, PCR products from preparations
containing 105, 104, 103,
102, 101, and 100 CFU/ml
respectively, after 6 h of enrichment; lanes 14 through 19, PCR
products from preparations containing 105, 104,
103, 102, 101, and 100
CFU/ml, respectively, after 9 h of enrichment; Lane 20, negative
control.
|
|
PCR detection with artificially contaminated tomatoes.
S. enterica serotype Montevideo was recovered from
inoculated tomatoes. Similar counts were obtained on BHI agar and
bismuth sulfite agar. Without enrichment, the detection limit was
105 CFU/tomato for surface-inoculated tomatoes (data
not shown). When samples were enriched in BHI broth at 37°C for
6 h, the detection limits were 102 and 103
CFU/tomato for Salmonella on and in tomatoes, respectively
(Fig. 4). Since the average weight of the
tomatoes was 75 g, the detection limits were equivalent to
101 and 102 CFU/g for Salmonella on
and in tomatoes, respectively.
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FIG. 4.
Use of HILA2-based PCR assay to detect S. enterica serotype Montevideo on and in artificially inoculated
tomatoes. Tomatoes were inoculated with different levels of S. enterica serotype Montevideo, and cells were then recovered and
enriched for 6 h. Crude DNA was prepared and subjected to PCR
amplification. (A) Detection of S. enterica serotype
Montevideo in tomatoes. (B) Detection of S. enterica
serotype Montevideo on tomato surfaces. Lane 1, 100-bp DNA ladders;
lanes 2 to 7, PCR products amplified from preparations containing
105, 104, 103, 102,
101, and 100 CFU/tomato, respectively; lane 8, negative control.
|
|
| |
DISCUSSION |
Various sets of primers for PCR detection of salmonellae have been
described previously (14, 18, 19, 24, 29, 30, 31). Gooding
and Chaudary (17) recently conducted a comparison study of
primers with different test panels of Salmonella and non-Salmonella strains and different PCR conditions and
found that there were variations with regard to the specificities
of the primers. The results of our study indicate that the
HILA2 primer set is very specific for Salmonella
strains, whereas the HILA1, SIRA1, and SIRA2 primers generated
false-positive results for non-Salmonella strains.
The lack of specificity of SIRA primers may be explained by the roles
of sirA genes in the pathogenicity of Salmonella.
It has been reported that all serovars of S. enterica encode
a type III protein secretion system within a pathogenicity island
(SPI1) at centisome 63 on the chromosome and that this system is
essential for pathogenicity (15). HilA is a transcriptional
regulator of the ompR-toxR family encoded within SPI1 and
controls expression of other SPI1 genes (6).
sirA, located outside SPI1, is an activator of
hilA (21), which is also known to be a global
regulator of several other genes mediating enteropathogenesis
(2). In addition, sirA appears to be a
housekeeping regulator that has been adapted to virulence gene
regulation in a variety of non-Salmonella bacteria, such as
Escherichia, Erwinia, Pseudomonas, and Vibrio strains (1). E. coli possesses a sirA
gene but does not contain hilA or SPI1 (2).
Therefore, it was understandable that the SIRA primer sets were not
specific for Salmonella.
Although both crude DNA and purified DNA worked well in PCR, the
detection limits were somewhat different. Crude DNA obtained after
cells were boiled could be detected in suspensions containing 105 CFU/ml, whereas the threshold of detection for
extracted DNA increased 1 log in a suspension containing
104 CFU/ml. Due to the longer preparation time, DNA was
prepared by boiling in further studies. If fresh produce is
contaminated with Salmonella, low numbers of cells would be
present, and thus enrichment would be required for detection.
Tomatoes, which have been implicated as sources of
Salmonella infection in several multistate outbreaks
(12; Wood et al., Epidemic Intelligence Service 40th
Annu. Conf.), were chosen as a model in this study. The HILA2 primer
set was found to detect S. enterica serotype Montevideo on
tomatoes. Fresh produce may contain a natural microflora that reflects
the production and harvest environments. Lactic acid bacteria and
Citrobacter, Enterobacter, Erwinia,
Pseudomonas, and Flavobacterium strains are
frequently associated with plant products (20). Ogunjimi et
al. (27) reported that endogenous polyphenol, which is
ubiquitous in plant products, interfered with immuno-PCR detection of
E. coli O157:H7. Moreover, ripe tomatoes have a pH range of
4.0 to 4.4. These factors may have an impact on PCR detection of
Salmonella in inoculated tomatoes. The results of this study
showed that in the presence of a natural biota, S. enterica
serotype Montevideo was recovered after centrifugation, and as little
as 101 and 102 CFU/g could be detected on and
in tomatoes, respectively, after 6 h of enrichment. The detection
limit for samples that were subjected to internal inoculation may have
been affected by sample treatment. Smaller populations of bacteria
(<102 CFU/g) may not be accessible to PCR due to physical
interference and thus would not be detected by the procedure used in
this study.
Our results demonstrate that HILA2 is very specific for
Salmonella. The PCR assay based on the HILA2 primer may also
be used to detect salmonellae on other fresh produce.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for Food
Safety and Quality Enhancement, Department of Food Science and
Technology, The University of Georgia, 1109 Experiment Street, Griffin,
GA 30223-1797. Phone: (770) 412-4740. Fax: (770) 229-3216. E-mail: lbeucha{at}cfsqe.griffin.peachnet.edu.
| |
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Applied and Environmental Microbiology, December 2000, p. 5248-5252, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
