-
2217
Journal of Food Protection, Vol. 71, No. 11, 2008, Pages
2217–2222
Identification of Salmonella Serotypes Isolated from
Cantaloupeand Chile Pepper Production Systems in Mexico by
PCR–Restriction Fragment Length Polymorphism
MIGUEL A. GALLEGOS-ROBLES,1 ALBERTO MORALES-LOREDO,2* GENOVEVA
ALVAREZ-OJEDA,3
ADRIÁN VEGA-P.,4 YAZMÍN CHEW-M.,4 SIXTO VELARDE,5 AND PINA
FRATAMICO6
1Facultad de Agricultura y Zootecnia, Universidad Juarez del
Estado de Durango, Venecia, Gomez Palacio, Durango, C.P. 35000,
México;2INIFAP, Campo Experimental General Terán, Carretera
Montemorelos-China Km. 31, General Terán, Nuevo León, C.P. 67400,
México;
3INIFAP, Campo Experimental Sur de Tamaulipas, Carretera
Tampico-Mante Km. 55, Estación Cuauhtémoc, México; 4INIFAP,
Campo ExperimentalLa Laguna, Blvd. Jose Santos Valdez No. 1200,
Col. Mariano Matamoros, C.P. 27440, México; 5INIFAP, Campo
Experimental Valle de Culiacán,
Carretera Culiacán-El dorado Km. 17.5, Culiacán, Sinaloa,
México; and 6U.S. Department of Agriculture, Agricultural Research
Service,Microbial Food Safety Research Unit, Eastern Regional
Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania
19038, USA
MS 08-107: Received 27 February 2008/Accepted 31 May 2008
ABSTRACT
A study was conducted in 2006 to determine the prevalence of
Salmonella on three cantaloupe farms in Matamoros,Coahuila, Mexico,
and on one farm that cultivates chile peppers var. Bell in
Culiacán, Sinaloa, Mexico. Samples from can-taloupe farms
consisted of cantaloupe rinses, irrigation water, water from
furrows in the field, and workers’ hands. Samplesfrom the chile
pepper farm consisted of rinses of chile peppers obtained at the
field, pepper rinses obtained at the packinghouse, and irrigation
water from the field. A total of 55 samples were obtained from both
production systems. Twelve and 10samples from the cantaloupe and
chile pepper production systems, respectively, tested positive for
Salmonella according to atraditional culture method. The difference
between the proportion of Salmonella-positive samples from the
cantaloupe pro-duction system (12 of 28 � 0.43) and the chile
pepper production system (10 of 27 � 0.37) was not statistically
significant(P � 0.05). A PCR–restriction fragment length
polymorphism (RFLP) method based on the fliC gene was used to
determinethe serotype of the isolates. Salmonella Typhimurium was
the only serotype found associated with the cantaloupe
productionsystem, whereas both Salmonella Typhimurium and
Enteritidis serotypes were found associated with the chile pepper
produc-tion system. Results showed that 91% (20 of 22) and 9% (2 of
22) of the isolates from both agricultural systems matchedwith the
Salmonella Typhimurium and Salmonella Enteritidis reference strain
restriction profiles, respectively. This studydemonstrates the
utility of the PCR-RFLP technique for determining the serotypes of
Salmonella isolates obtained fromcantaloupe and chile pepper
production systems.
The genus Salmonella comprises over 2,700 serotypesthat are
found in different hosts and environments and thatcan cause human
illness, including enteric fever, gastroen-teritis, and septicemia.
Salmonella infection has been as-sociated with the consumption of
contaminated fresh fruitsand vegetables, raw and undercooked
poultry, or other redmeat and poultry products (1, 13, 24). Most
studies in Mex-ico on Salmonella tend to corroborate the presence
or ab-sence of Salmonella in food and water and as a cause
ofoutbreaks; however, only a few studies have been per-formed
investigating the prevalence of different Salmonellaserotypes in
Mexico (6, 14). An important phenotypic char-acteristic of the
genus is the antigenic diversity in the fla-gellar antigens, which
is also observed at the genetic level(10).
The procedures used to identify Salmonella are labo-rious,
time-consuming, and require a number of biochem-ical and
serological tests to confirm presumptive isolates(25). On the other
hand, tests using molecular tools havebeen useful in reducing the
steps and the time needed for
* Author for correspondence. Tel and Fax: 52-81-83674487, Ext
132;E-mail: [email protected].
the detection, identification, and characterization of
specificpathogens. The fliC gene, encoding for the flagellin
protein,has been used as a target gene in assays to test the
geneticdiversity in Salmonella (10). The fliC gene has a
conservedterminal region and a variable central region, which
deter-mines the antigenic specificity (4, 15). The goals of
thisstudy were to determine the prevalence and sources of
Sal-monella in cantaloupe and chile pepper production systemsin
Mexico and to apply a PCR–restriction fragment lengthpolymorphism
(RFLP) method based on the fliC gene toidentify the serotypes of
the Salmonella isolates.
MATERIALS AND METHODS
Samples from cantaloupe and chile pepper farms. Sam-ples from
cantaloupe production systems were obtained at threefarms (fields)
in Matamoros, Coahuila, Mexico, during June 2006.The samples
consisted of cantaloupe rinses (five composite sam-ples per farm,
125 ml per composite sample), water obtained froman irrigation
channel that irrigated the three farms (four samples,25 ml per
sample), water from furrows in fields (one sample perfarm, 25 ml
per sample), and samples from workers’ hands (sixsamples from one
farm, 1 cotton swab per worker). Samples fromthe chile pepper var.
Bell production system were obtained fromonly one farm located in
Culiacan, Sinaloa, Mexico, during April
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J. Food Prot., Vol. 71, No. 112218 GALLEGOS-ROBLES ET AL.
TABLE 1. Salmonella reference strains used in this study
andisolates from cantaloupe and chile pepper production systems
Salmonella serotype Origin/sourcea
Reference strains
1 Paratyphi A ATCC 91502 Enteritidis ATCC 130763 Typhimurium
ATCC 133114 Kentucky IBT-UNAM5 Typhi IBT-UNAM6 Worthington
IBT-UNAM7 Stanley IBT-UNAM8 Typhimurium IBT-UNAM
Serotype determined by PCR-RFLPCantaloupe production system
9 Typhimurium Matamoros, Coahuila/fruit rinse10 Typhimurium
Matamoros, Coahuila/fruit rinse11 Typhimurium Matamoros,
Coahuila/fruit rinse17 Typhimurium Matamoros, Coahuila/fruit
rinse18 Typhimurium Matamoros, Coahuila/fruit rinse19 Typhimurium
Matamoros, Coahuila/fruit rinse12 Typhimurium Matamoros,
Coahuila/water from channel13 Typhimurium Matamoros, Coahuila/water
from channel14 Typhimurium Matamoros, Coahuila/water from furrow15
Typhimurium Matamoros, Coahuila/water from furrow16 Typhimurium
Matamoros, Coahuila/water from furrow20 Typhimurium Matamoros,
Coahuila/workers’ hands
Chili pepper production system
21 Typhimurium Culiacán, Sinaloa/water from channel22
Typhimurium Culiacán, Sinaloa/water from channel23 Typhimurium
Culiacán, Sinaloa/water from channel24 Typhimurium Culiacán,
Sinaloa/water from channel25 Typhimurium Culiacán, Sinaloa/water
from channel26 Enteritidis Culiacán, Sinaloa/water from channel27
Enteritidis Culiacán, Sinaloa/fruit rinse28 Typhimurium Culiacán,
Sinaloa/fruit rinse29 Typhimurium Culiacán, Sinaloa/fruit rinse30
Typhimurium Culiacán, Sinaloa/fruit at packing
a ATCC, American Type Culture Collection; IBT-UNAM, Insti-tuto
de Biotecnologı́a Universidad Nacional Autónoma de Mé-xico.
2006 and consisted of rinses of chile fruits obtained at the
field(five composite samples, 125 ml per composite sample), fruit
rins-es obtained at the packing house (eight samples, 25 ml per
sam-ple), and water from the irrigation channel (14 samples, 25 ml
persample). A total of 55 samples were obtained from both
produc-tion systems. Cantaloupe and chile pepper rinses were
obtained atboth production systems from five representative points
at eachfarm, and at each point, five fruits were taken randomly
within aradius of 3 m. Each fruit was washed individually by
placing thefruit into a sterile Whirl-Pak bag (Nasco, Modesto,
Calif.) con-taining 25 ml of 0.1% buffered peptone water (Becton
Dickinson,Sparks, Md.) with a different sterile pair of gloves for
each fruit.The fruit was washed by shaking and mixing the bag for
at least2 min. The washings from the five cantaloupes obtained from
thesame point were combined into a single glass bottle (125 ml
percomposite sample). Samples from water sources were placed
di-rectly into sterile glass bottles (25 ml per sample). Samples
fromhands of workers in the field were obtained by rubbing the
handswith a sterile cotton swab (Dequinsa, D.F. México) moistened
in0.85% saline solution and then placing the cotton swab into a
tubewith 5 ml of saline solution. Fruit rinses obtained at the
packinghouse were obtained in a similar way as the rinses in the
field,except each fruit constituted an individual sample. The
sampleswere immediately transported in a cooler containing ice to
thelaboratory and processed within 24 h. Each sample was mixed
byshaking; 1.0 ml was removed and added to 9.0 ml of
bufferedpeptone water and then incubated at 35�C for 24 h.
Enrichmentand microbiological analyses were performed according to
themethod described in the U.S. Department of
Agriculture–FoodSafety and Inspection Service Microbiology
Laboratory Guide-book (23). Briefly, 0.5 � 0.05 ml from the
buffered peptone waterpreenrichment was transferred into 10 ml of
tetrathionate brothand 0.1 � 0.02 ml into 10 ml of modified
Rappaport-Vassiliadisbroth, which were then incubated at 42 � 0.5�C
for 22 to 24 h.Next, both enrichments were streaked onto brilliant
green sulfaand double-modified lysine iron agar plates and
incubated at 35� 2�C for 18 to 24 h. Plates were then examined for
the presenceof colonies meeting the description of suspect
Salmonella colo-nies.
Salmonella strains. The control strains and isolates obtainedin
this study are listed in Table 1. Salmonella enterica
serotypesTyphimurium (American Type Culture Collection [ATCC]
13311,Manassas, Va.), Enteritidis (ATCC 13076), and Paratyphi
A(ATCC 9150) were used as positive controls. Salmonella
entericaserotypes Kentucky, Stanley, Typhi, Typhimurium, and
Worthing-ton, kindly provided by Edmundo Calva (Instituto de
Biotecno-logı́a, Universidad Nacional Autónoma de Mexico,
Cuernavaca,Morelos, México) also were included in the study as
positive con-trols. Biochemical tests and serotyping of all of the
above-men-tioned strains were performed at the institution from
which theywere obtained. In addition, 22 isolates obtained from the
two ag-ricultural production systems that were not serotyped were
in-cluded, for a total of 30 isolates subjected to PCR-RFLP
analysis.
Data analyses. The test of difference between two
binomialproportions (19) was performed to see whether the
difference be-tween the proportions of positive samples from the
agriculturalproduction systems was significant.
DNA extraction. Each isolate was streaked onto Trypticasesoy
agar plates and incubated at 35�C for 24 h. Colonies fromeach agar
plate were removed with a loop and then suspended in3 ml of
buffered peptone water and incubated at 35�C for 24 h.The entire 3
ml of the culture was centrifuged (Sigma 1-15K,
Sigma Laborzentrifugen, Gottingen, Germany) three times, 1
mleach time, at 3000 � g for 5 min in 1.5-ml Eppendorf tubes.DNA
extraction was performed on the resulting cell pellet ac-cording to
the CTAB method, but omitting the use of polyvinyl-pyrrolidone and
�-mercaptoethanol (5). The DNA samples werestored at �20�C until
used.
PCR amplification of the fliC gene. Primers
ABMS1(5-GCACAAGTCATTAATACAAACAGCC-3) and
ABMS2(5-TTAACGCAGTAAAGAGAGGACG-3) described by Daugaet al. (4) were
modified (by adding the bases that are underlined)on the basis of
analysis with Amplify 1.2 software (University ofWisconsin,
Madison) and used to amplify a fragment of approx-imately 1.5 kb
from the fliC gene in each Salmonella strain. PCRamplification of
the target sequence was performed in a PCR Ex-press thermal cycler
(Thermo Hybaid, Ashford, Middlesex, UK).The PCR mixture contained
25 pmol of each of the primers, 250
M of a mix of deoxynucleoside triphosphates (Invitrogen,
Car-slbad, Calif.), 1 mM MgCl2, 1� reaction buffer (200 mM
Tris-HCl, pH 8.0, 500 mM KCl), 2.5 U Taq DNA polymerase
(Bioline,
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J. Food Prot., Vol. 71, No. 11 IDENTIFICATION OF SALMONELLA
SEROTYPES BY PCR-RFLP 2219
TABLE 2. Samples positive for Salmonella from the cantaloupe and
chile pepper production systems showing the number and type
ofsamples and serotype of isolatesa
Type of sample
No. of positive samples/total analyzed(proportion)
Cantaloupe Chile pepper
Salmonella serotype found (no. of positive samples containingthe
serotype/total analyzed)
Cantaloupe Chile pepper
Fruit rinses 6/15 (0.4) 3/5 (0.6) Typhimurium (6/15) Typhimurium
(2/5), Enteritidis (1/5)Water from irrigation channel 2/4 (0.5)
6/14 (0.43) Typhimurium (2/4) Typhimurium (5/14), Enteritidis
(1/14)Water from farm 3/3 (1.0) — Typhimurium (3/3)Workers’ hands
1/6 (0.16) — Typhimurium (1/6)Fruit rinses at packing house — 1/8
(0.12) — Typhimurium (1/8)
Total 12/28 (0.43) 10/27 (0.37) Typhimurium (12/28) Typhimurium,
(8/27), Enteritidis (2/27)
a —, none of these types of samples were obtained.
Taunton, Mass.), 100 ng of DNA template, and deionized waterfor
a final volume of 25 l. The reaction mixture was subjectedto PCR
under the following conditions: heat denaturation at 95�Cfor 1 min
and then 35 cycles of heat denaturation at 94�C for 30s, primer
annealing at 60�C for 30 s, and DNA extension at 72�Cfor 30 s.
After the last cycle, the PCR tubes were maintained at72�C for 10
min to complete synthesis of all strands. Five micro-liters of each
PCR reaction was loaded onto a 1.5% agarose gel(Promega, Madison,
Wis.), and electrophoresis was performedwith the use of 1� SB
buffer (0.2 M NaOH, 0.7278 M boricacid). The gel was stained with
ethidium bromide, examined witha UV transilluminator (Spectroline
Transilluminator, model 7C-254R, Electronics Corporation, Westbury,
N.Y.), and photo-graphed with a Polaroid camera (adapted with UV
filter, filmA667). A 250-bp ladder (Invitrogen) was used as a
molecularweight standard.
RFLP analysis. For the PCR-RFLP analysis, the
unpurifiedamplified PCR product of the fliC gene was cleaved with
Sau3AI(Promega) according to the manufacturer’s instructions. The
di-gestion reaction consisted of 10 l of PCR product, 2 l of
10�reaction buffer B (60 mM Tris-HCl [pH 7.5] 500 mM NaCl, 60mM
MgCl2, 10 mM dithiothreitol), 0.2 l of acetylated BSA (10
g/l), 5 U of enzyme, and deionized water for a final volumeof 20
l. The reaction was gently vortexed and then incubated at37�C for 1
h. After incubation, 6 l of sample was mixed with 3
l of loading buffer (0.25% bromophenol blue, 0.25% xylene
cy-anole, 30% glycerol) and electrophoresed on a 10% acrylamidegel
for 2.5 h at 100 V in 1� Tris-borate-EDTA buffer. The 100-bp
Hyperladder (Bioline) was used as the molecular weight stan-dard
for determining the molecular weight of the restriction frag-ments.
Gels were stained and photographed as described above.The degree of
variability between two strains was determined onthe basis of the
Dice coefficient, and a dendrogram was made bythe unweighted pair
group method with arithmetic mean (22). TheSPSS (Statistical
Product for Service Solutions, v 10.0, SPSS Inc.,Chicago, Ill.)
statistical package was used for the analyses.
RESULTS AND DISCUSSION
Samples positive for Salmonella. The proportions ofpositive
samples, obtained by dividing the number of sam-ples positive for
Salmonella by the total number of samples,were 0.43 and 0.37 from
the cantaloupe and chile pepperproduction systems, respectively,
and the observed differ-ence was not statistically significant (P �
0.05). Althoughthe number of samples obtained from the cantaloupe
farmswas quite small, the observed differences among the three
farms were not statistically significant (P � 0.05). A totalof
22 Salmonella isolates were obtained from the two ag-ricultural
production systems: 12 from the cantaloupe farmand 10 from the
chile pepper farm. From the cantaloupeproduction system, at least
one sample from each sourcewas positive for Salmonella, and water
sources had higherproportions of positive samples (5 of 7) (P �
0.05) com-pared with the other samples (Table 2). From the chile
pep-per production system, a higher number of positive samplescame
from fruit rinses at the field and water from the irri-gation
channel. These results demonstrate that water fromthe irrigation
channel was an important source of Salmo-nella contamination.
Investigations examining the environ-mental sources of Salmonella
contamination indicate thatwater is an important source,
particularly irrigation watercontaining manure, feces from animal
wildlife, or sewageeffluent (9, 20). A likely reason why Salmonella
was foundin water samples is that the water used for irrigation of
thecantaloupe farms originated from the Rio Nazas and flowedthrough
open channels without any protection against con-tact with animals.
In addition, the cantaloupe farms wereirrigated from the same
channels used to irrigate foragecrops with sewage. Castillo et al.
(2) stated that Salmonellaand Escherichia coli were frequently
detected in water usedfor irrigation of cantaloupe farms in South
Texas becausethe water came from the Rio Grande. From the chile
pepperfarm, 6 of 14 irrigation water samples were positive, and 3of
5 fruit rinse samples were positive. It is possible thatinsects or
birds could also have transmitted Salmonella tothe fruits. Moore et
al. (16) mentioned the possibility thatinsects belonging to the
genus Chironomus were direct orindirect vectors of enteric bacteria
for water and foods. Onthe other hand, Sela et al. (21) stated that
direct contactwith manure-contaminated soil or dust might be a
sourceof preharvest contamination, and indirect sources of
con-tamination could also be due to trophic interactions
betweenfruit and plant foragers like birds, mammals, and
insects.The lowest numbers of positive samples were from work-ers’
hands at the cantaloupe farm and from rinses of chilepeppers
obtained at the chile pepper packing house. Con-tamination in these
samples could be due to worker’s poorhygiene, cross-contamination
from fruit to worker, as wellas poor quality of the water used in
the packing house to
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J. Food Prot., Vol. 71, No. 112220 GALLEGOS-ROBLES ET AL.
FIGURE 1. PCR products obtained using ABMS1 and ABMS2primers.
(A) Lane 1, Salmonella Paratyphi A (ATCC 9150); lane2, Salmonella
Enteritidis (ATCC 13076); lane 3, Salmonella Ty-phimurium (ATCC
13311), lane 4, negative control. (B) Lane 1,Salmonella Kentucky;
lane 2, Salmonella Typhi; lane 3, Salmo-nella Worthington; lane 4,
Salmonella Stanley; lane 5, SalmonellaTyphimurium; lane 6, negative
control; lanes M, 250-bp ladder(Invitrogen).
wash the fruit. The Centers for Disease Control and Pre-vention
reported that in outbreaks that occurred in 2001 and2002 associated
with the consumption of cantaloupes fromMexico contaminated with
Salmonella Poona, possiblesources of contamination included
irrigation of fields withwater contaminated with sewage, processing
(cleaning andcooling) produce with Salmonella-contaminated water,
poorhygienic practices of workers who harvested and processedthe
cantaloupes, pests in packing facilities, and inadequatecleaning
and sanitizing of equipment that came in contactwith the
cantaloupes (3).
PCR amplification. The PCR product from the refer-ence strains
(Fig. 1A) and from the different serotypes (Fig.1B) was of the
expected size on the basis of the simulatedPCR product obtained
with Amplify 1.2 software. As an-ticipated, the sizes were 1,487 bp
for Salmonella ParatyphiA and Salmonella Typhimurium, 1,517 bp for
SalmonellaEnteritidis (Fig. 1A), and 1,520 bp for Salmonella
Typhi(Fig. 1B). Figure 1B shows the PCR products of varioussizes
from the different Salmonella serotypes as evidenceof the genetic
variability of the fliC gene. The PCR productsof Salmonella
isolates obtained from both production sys-tems were approximately
1.5 kb in size. PCR with the prim-er set described by Kilger and
Grimont (10) for amplifi-cation of the fliC gene generated products
of two differentsizes: 1.24 kb for serotype Typhi and 1.5 kb for
all otherserotypes. The difference in the size of the PCR
productsobtained for the fliC gene can be accounted for by the
var-iability of the central region of the open reading frame ofthis
gene (8).
PCR-RFLP analysis. PCR-RFLP was carried out on30 strains, 8
belonging to 7 different Salmonella serotypesand 22 of unknown
serotype isolated in this study (oneisolate from each positive
sample) (Table 1). PCR-RFLPanalyses with Sau3AI on Salmonella
strains of seven dif-ferent known serotypes yielded seven distinct
restrictionprofiles for the fliC gene (Fig. 2A), demonstrating that
fliCis a suitable target gene for discriminating among Salmo-nella
serotypes by PCR-RFLP. Using PCR-RFLP on thegroEL gene amplicon of
Salmonella digested with HaeIII,Nair et al. (18) found a low
discriminatory capacity, be-cause only three different profiles
were obtained, and dif-ferent serotypes of Salmonella shared the
same restrictionprofile. However, other researchers have found good
dis-criminatory ability with the fliC and fljB genes,
particularlywith the use of a double digestion (4, 8). A DNA
sequence–based approach examining the Salmonella flagellin fliCgene
revealed the existence of two groups: the g-complex,which included
Salmonella Enteritidis and nonmotilestrains, and the non–g-complex,
which included motilestrains such as Salmonella Typhimurium (17).
Results ofthis study indicated that there was a high level of
sequencehomology between fliC genes of g-complex strains, and
thatthe genetic basis for distinct antigens in this group of
se-quences can be due to a single amino acid substitution.Also, it
has been reported that differences in the fliC genesequence coding
different antigenic phase 1 types could bedue to 1 to 44 nucleotide
substitutions, some of which resultin changes in the amino acid
sequence of the flagellin pro-tein (7).
Comparing the restriction profiles obtained with theSalmonella
isolates from the cantaloupe and chile pepperproduction systems
(Fig. 2B and 2C, respectively) withthose of the reference strains,
all 12 isolates obtained fromthe cantaloupe production system had
the Salmonella Ty-phimurium restriction profile. Six isolates were
from rinsesof cantaloupe surfaces (two from each orchard), five
werefrom water used for irrigation, and one was from the handsof a
field worker. From the chile pepper production system,eight
isolates were also identified as Salmonella Typhimu-rium: five from
the water used for irrigation, two from chilepeppers, and one from
chile peppers at the packing house.In addition, a second Salmonella
restriction profile wasfound that matched that of Salmonella
Enteritidis. It wasfound in two isolates: one from an irrigation
water sampleand one from a chile pepper rinse sample.
Results from the PCR-RFLP analyses based on the fliCgene showed
that 91% (20 of 22) and 9% (2 of 22) of theisolates from both
agricultural systems matched with theSalmonella Typhimurium and
Salmonella Enteritidis re-striction profiles, respectively, and
these were the only twoserotypes found in both agricultural
systems. This is inagreement with previous reports that indicate
that Salmo-nella Typhimurium and Salmonella Enteritidis are the
se-rotypes most commonly isolated from outbreaks of
humansalmonellosis linked to the consumption of contaminatedanimal
and vegetable foods (11, 12). It is important that50% of the
isolates were obtained from water sources,which points out the risk
associated with water used in
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J. Food Prot., Vol. 71, No. 11 IDENTIFICATION OF SALMONELLA
SEROTYPES BY PCR-RFLP 2221
FIGURE 2. Restriction profiles of the Salmonella fliC gene
obtained with Sau3AI endonuclease digestion. (A) Lane 1,
SalmonellaKentucky; lane 2, Salmonella Typhi; lane 3, Salmonella
Worthington; lane 4, Salmonella Stanley; lane 5, Salmonella
Paratyphi A(ATCC 9150); lane 6, Salmonella Typhimurium (ATCC
13311); lane 7, Salmonella Enteritidis (ATCC 13076). (B) Isolates
obtained atthe cantaloupe production system. Lanes 1 through 7,
restriction profile of Salmonella Typhimurium. (C) Isolates
obtained from thechile pepper production system. Lanes 1 through 5
and 8 through 10, restriction profile of Salmonella Typhimurium;
lanes 6 and 7,restriction profile of Salmonella Enteritidis; lanes
M, 100-bp ladder (Invitrogen).
cantaloupe and chile pepper production systems. These re-sults
partially agree with those obtained by Castillo et al.(2), who
reported that most isolates they recovered werefrom water samples;
however, the Salmonella serovars iso-lated from water were
different from those isolated fromthe melons. It is important that
Salmonella Poona, which isa serotype that often causes outbreaks of
salmonellosis inthe United States associated with the consumption
of mel-ons from Mexico, was not found in this study, because
theexpected restriction profile for the fliC gene of this
serotype(simulated with DNA Straider 1.2, GeneBank
accessionAY353467 for the fliC gene of Salmonella Poona) was
notobserved among the restriction profiles obtained in thisstudy.
This provides some evidence that this serotype mightbe present only
at melon production areas in southeasternMexico, where iguanas,
which are considered a reservoirfor this serotype, are found (3).
Further studies are neededto determine this possibility.
Although the number of samples analyzed in this studywas small,
it was demonstrated that irrigation water couldbe an important
source of contamination of produce by Sal-monella Typhimurium and
Salmonella Enteritidis. Salmo-nella might have been transmitted by
direct contact of thewater with the melons or by contact of the
melons or thewater to other parts of the production systems,
includingthe workers. It is also possible that contamination from
illpersonnel during the handling of the fruit or
contaminationthrough vectors, including insects and birds, could
have oc-curred; however, these possibilities were not
investigatedhere. The results of this investigation suggest that in
pro-duction systems in which Good Agricultural Practices arenot in
place, as in the farms tested in this study, contami-nation with
Salmonella can occur, representing a health riskfor the farmers and
the consumers of the contaminated pro-duce. Although only a small
number of strains were tested,this study demonstrated the utility
of the PCR-RFLP tech-nique for determining the serotypes of
Salmonella isolatesobtained from cantaloupe and chile pepper
production sys-tems by comparison to restriction profiles of known
refer-
ence strains. The method is rapid, simple, and reproducibleand
can potentially be applied for identification of isolatesobtained
from other production systems. More extensivestudies need to be
performed examining a larger number offarms and samples to
determine the prevalence of Salmo-nella in agricultural production
systems. Typing of isolatesby pulsed-field gel electrophoresis
might be useful forsource tracking of Salmonella on the farms.
Production sys-tems with Good Agricultural Practices and Good
Manufac-turing Practices in place should be compared with those
thatdo not use Good Agricultural Practices and Good Manu-facturing
Practices to determine the efficacy of such prac-tices for the
prevention of Salmonella contamination.
ACKNOWLEDGMENT
The Consejo Nacional de Ciencia y Tecnologı́a is acknowledged
forfunding this research.
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________________________________________________________Revista
Científica, FCV-LUZ / Vol. XIX, Nº 2, 139 - 146, 2009
CARACTERIZACIÓN DE AISLADOS DE Escherichia coli O157:H7EN
CANALES DE BOVINOS Y PORCINOS MEDIANTE PCR.
Characterization of Escherichia coli O157:H7 Isolates Obtained
from Bovine and PorcineCarcasses.
Miguel Gallegos1, Alberto Morales2*, Genoveva Álvarez 2, Jesús
Vásquez 3, Lilia Morales4, Irma Martínez 4
y Jesús Maldonado5
1Facultad de Agricultura y Zootecnia, Universidad Juárez del
Estado de Durango. 2Instituto Nacional de Investigaciones
Forestales,
Agrícolas y Pecuarias (INIFAP) hasta 31 de diciembre del 2007,
actualmente Consorcio Técnico del Noreste, A. C. UGRNL.
Teléfono y Fax: 01 81 83674487 Ext. 132. Km 4.5 carretera a
Reynosa, Guadalupe, N. L. México.3Facultad de Ciencias Químicas,
UJED. 4Facultad de Ciencias Biológicas, UANL. 5Laboratorio de
Diagnóstico Molecular,
Núcleo “Héctor Ochoa Zuleta”, Universidad Centroccidental
“Lisandro Alvarado”, Venezuela.
*E-mail: [email protected]
RESUMEN
Muchos de los brotes causados por Escherichia coli O157:H7se han
asociado al consumo de carne bovina mal cocida, perotambién se ha
reportado su presencia en la carne de otros ani-males domésticos.
En México existe poca información sobre lapresencia de este
patógeno en canales de res y de cerdo. Elobjetivo de este estudio
fue determinar la presencia de E. coliO157:H7 en canales de res y
cerdo y su caracterización me-diante PCR. De un total de 18
aislados, 12 fueron positivas porPCR para los genes rfbE y fliC que
determinan el serotipoO157:H7. De estos 12, uno de canal de res y
tres de canalesde cerdo fueron positivos por PCR para los genes
stx1, stx2 yeaeA, por lo que fueron considerados como
enterohemorrági-cos. Las diferencias encontradas en el número de
canales po-sitivas para los genes caracterizados no fueron
estadística-mente significativas, y los resultados señalan que E.
coliO157:H7 puede ser encontrada en ambos tipos de canal,
re-presentando un riesgo para la salud, por lo que se deben to-mar
medidas más estrictas de higiene y manejo para evitarque canales
que no cumplan con el carácter de inocuidad lle-guen a los
consumidores finales.
Palabras clave: Bovinos, cerdos, E. coli O157:H7,
enterohe-morrágica, caracterización molecular, PCR.
ABSTRACT
Many Escherichia coli O157:H7 outbreaks have been associ-ated to
consumption of undercooked beef, but the presencehas also been
reported in the meat of other domestic animals.In Mexico, little
information exists on the presence of thispathogen in bovine and
pork carcasses. The objective of thisstudy was to determine the
presence and/or absence of E. coliserotype O157:H7 in bovine and
pork carcasses and theircharacterization by means of PCR. Of 18
isolates, 12 werepositive by PCR to rfbE and fliC genes, which
determineO157:H7 serotype. Of these 12, one from bovine carcass
andthree of pork carcasses were positive by PCR to stx1, stx2
andeaeA genes, therefore, they were considered enterohemor-rhagic
strains. The differences found in the positive carcassnumber to any
of the genes were not statistically significant.The results show
that E. coli O157:H7 could be found in bothcarcasses types,
representing a risk for the health, so strict hy-gienic and
handling measures should be taken in order toavoid that carcasses
which do not fulfill the food safety aspectmight arrive to the
final consumers.
Key words: Bovine and pork carcasses, E. coli O157:H7,
en-terohemorrhagic, molecular characterization, PCR.
INTRODUCCIÓN
Escherichia coli O157:H7 ha emergido como un patóge-no
transmitido por alimentos y es considerado de importanciaen salud
pública, ya que está implicado en brotes de colitis he-
139
Recibido: 22 / 02 / 2007. Aceptado: 08 / 03 / 2008.
-
morrágica y posible aparición posterior del síndrome
urémicohemolítico (SUH) [19]. Una característica de E. coli
O157:H7es el bajo número de células requeridas para desarrollar la
en-fermedad de 10 a 100 células [11] por lo que la no detecciónpor
los métodos tradicionales microbiológicos no es certeza nisinónimo
de seguridad del alimento. E. coli O157:H7 puedeestar presente en
una gran variedad de animales silvestres ydomésticos entre los
cuales se encuentran bovinos (Bos tau-rus-indicus), porcinos (Sus
scrofa domestica) y ovinos (Ovisaries) [18], siendo principalmente
los rumiantes y sus heces fe-cales un reservorio natural de este
patógeno [17, 20]. Se hareportado que la transmisión de E. coli
O157:H7 a los huma-nos en forma directa o indirecta puede ser por
contaminaciónde los alimentos a partir de material fecal, agua
contaminada,y contacto con personas o animales enfermos [4]. Por
otrolado, microorganismos patógenos para el humano como E.
coliO157:H7, que habita naturalmente en el tracto digestivo
delganado bovino, puede eventualmente contaminar la canal du-rante
el proceso de evisceración o en el manejo posterior de lamisma
[25]. Aunque el serotipo de E. coli O157:H7 es determi-nado por los
genes rfbE y fliC que codifican respectivamentepara la biosíntesis
del lipopolisacárido O157 y la flagelina, loque a su vez determina
respectivamente los antígenos O y H,la patogenicidad se debe a la
expresión de varios genes quecodifican para factores de virulencia.
Entre estos se encuen-tran los genes stx1 y stx2 que codifican para
las verotoxinas 1y 2, respectivamente, los cuales se encuentran en
un bacterió-fago integrado al cromosoma bacteriano y que han sido
aso-ciados al desarrollo del SUH, particularmente stx2 [21]. Otrode
los genes de virulencia localizado en el locus LEE (locus deefusión
en enterocitos) es el gen eaeA que codifica para la inti-mina y que
es necesario para el proceso de la adherencia ínti-ma a las células
epiteliales del intestino humano [12, 20, 22].En México existen
algunos reportes de la presencia de E. coliO157:H7 en muestras
comerciales de carne de res, pero no sedefine si la carne venía
contaminada desde el matadero o secontaminó en el manejo posterior
[6], y siendo México un paíscon una producción promedio en los
últimos cinco años de1.473.650 t de carne de bovino y de 1.050.311
t de carne decerdo [24] y un volumen de exportación combinado de
ambostipos de carne de 38.677,2 t [23], es importante determinar
lacalidad sanitaria de la canal al momento de salir del matadero.El
objetivo del presente estudio consistió en caracterizar cepasde E.
coli del serotipo O157:H7 aisladas de canales de bovi-nos y
porcinos en plantas procesadoras de productos cárnicosde la Comarca
Lagunera, México.
MATERIALES Y MÉTODOS
Diseño del estudio
El estudio fue de tipo longitudinal prospectivo y el perío-do
comprendió de febrero a marzo del 2004. Se seleccionarondos plantas
procesadoras de productos cárnicos, una de cerdo
(I) y una de bovino (II) de la Comarca Lagunera, México.
Losmuestreos se realizaron en dos fechas para cada
planta,20/02/2004 y 15/03/2004 para la planta I y 02/03/2004
y24/03/2004 para la planta II. A partir de estos se obtuvieron
losaislados de E. coli.
Obtención de las muestras
La toma y manejo de las muestras se realizó conforme alo
especificado por la Norma Oficial Mexicana NOM-109SSA1-1994 y con
modificaciones a lo propuesto por Gill y Jo-nes [15]. Las canales
se muestrearon inmediatamente a suarribo al área de inspección y
sellado (15 y 10 minutos en pro-medio después del inicio del
sacrificio del animal hasta la tomade las muestras, respectivamente
para canal de bovino y cer-do). En cada planta procesadora se
procedió a la selección de15 canales al azar, para el análisis de
superficies vivas, de lascuales se recolectaron las muestras con
esponja estéril NAS-CO®, Whirl-Pak, Speci-Sponge, B01324WA (5x10x1
cm) (Nas-co EUA) en tres sitios diferentes: falda (sitio A),
costado (sitioB) y cuello (sitio C), para un total de 45 muestras
por planta.De las 15 muestras de cada sitio de la canal (A, B, C)
se for-maron tres grupos de cinco muestras cada uno, para
tenernueve muestras compuestas por planta, lo que dio un
tamañofinal de 18 muestras compuestas.
Aislamiento y confirmación de E. coli O157:H7en muestras
compuestas
Cada muestra compuesta se pasó a enriquecimiento encaldo Reveal
(NEOGEN®, EUA) y se incubó durante 8 h a42°C, luego de este tiempo
se tomaron 120 µL y se deposita-ron sobre la placa de lectura del
kit. Después de 15 minutos serealizaron las lecturas y de las
muestras presuntivas se tomóuna asada y se estrío en medio
cromogénico CHROmagar®
O157 (CHROmagar, Francia). Las colonias características
seconfirmaron con pruebas bioquímicas utilizando el API® 20
E(Biomereux, Francia) y el serotipo utilizando un kit de
serologíaDifco™ E. coli Antiserum (DIFCO, EUA). En las muestras
quese confirmó la presencia de E. coli O157:H7 se procedió a
rea-lizar la obtención de ADN para la caracterización
molecularmediante la Reacción en Cadena de la Polimerasa (PCR).
Cepa de referencia, condiciones y medio de cultivo
La cepa de referencia de E. coli O157:H7 que se usócomo control
positivo en los ensayos de PCR fue proporciona-da por el Centro
para el Control y la Prevención de Enferme-dades (CDC) a través del
Centro Nacional de Servicios deConstatación en Salud Animal
(CENAPA), México. La cepa fueactivada en caldo infusión cerebro
corazón a 35°C por 24 h.
Extracción del ADN
Los aislados de E. coli O157:H7 se incubaron en caldoinfusión
cerebro corazón por 24 h a 37°C. Después de la incu-bación y a
partir de este medio se tomó tres veces 1 mL y se
140
Caracterización de aislados de E. coli O157:H7 en canales de
bovinos y porcinos mediante PCR / Gallegos, M. y
col.__________________
-
centrifugó a 3000 rpm durante 1 min (Centrifuga Sigma
1-15K,Alemania) para formar una pastilla en tubos Eppendorf de
1,5mL y hacer la extracción de ADN con el método CTAB [7]
peroomitiendo el uso de polivinilpirrolidona y
2�-mercaptoetanol.
Reacciones de PCR
Las reacciones de PCR se realizaron utilizando los
oligo-nucleótidos mencionados en la TABLA I. Primero se realizóuna
reacción para amplificar simultáneamente dos
fragmentoscorrespondientes a los genes rfbE y fliC [5, 13]. En los
aisla-dos que resultaron positivos para los genes anteriores, se
rea-lizaron reacciones por separado para amplificar un fragmentoen
cada uno de los siguientes genes: stx1, stx2 y eaeA [1, 14].Las
reacciones de PCR se realizaron en volúmenes de 25 µL,utilizando 1
µL (25 pmoles) de cada uno de los oligonucleóti-dos, 2,5 µL de los
dNTP´s (200 µM) (GIBCO-BRL), 0,5 µL deMgCl2 (1,5 mM), 2,5 µL de
buffer para PCR (10X) (200 mMTris-HCl pH 8,0, 500 mM KCl), 2,5
unidades de la enzimaTaq-DNA polimerasa (Bioline) y 1 µL de ADN
(100 ng). El con-trol negativo de la PCR tenía los mismos
ingredientes, exceptoel ADN, sustituyéndose este volumen con agua
miliQ estéril.La amplificación se llevó a cabo en un termociclador
PCR Ex-press (ThermoHybaid, Reino Unido) y las condiciones de
corri-da para los genes rfbE y fliC fueron de un ciclo de 1 min
a95°C, seguido de 35 ciclos de tres pasos: desnaturalización a95°C
por 30 s, alineamiento a 66°C por 30 s y una extensión a72°C por 75
s, con una extensión final de 10 min a 72°C. Paralos genes stx1 y
stx2 se modificó el paso de alineamiento a58°C durante 30 s. Para
el gen eaeA se modificó el paso dealineamiento a 64°C por 30 s. Las
demás temperaturas y tiem-pos fueron iguales. Los productos de PCR
fueron visualizadosen geles de agarosa al 1,5% teñidos con bromuro
de etidio(0,5 µg mL–1), y visualizados en un transiluminador de luz
UV(Spectroline, EUA). Posteriormente fueron fotografiados conuna
cámara polaroid (película A667, EUA) adaptada con filtropara luz
ultravioleta.
Prueba estadística
Se usó la prueba de Ji cuadrado [3] para probar si las
di-ferencias observadas en el número de canales positivas al
pa-tógeno de cerdo y de bovino eran estadísticamente
significati-vas.
RESULTADOS Y DISCUSIÓN
De un total de 18 muestras (nueve de cerdo y nueve deres), seis
de cerdo (66,66%) y seis de bovino (66,66%) fueronpositivas por PCR
para la presencia de E. coli serotipoO157:H7, demostrándose así que
este patógeno puede estarpresente en ambos tipos de canal (TABLA II
y FIG. 1). E. coliO157:H7 se ha encontrado en una gran variedad de
alimentos,tanto de origen vegetal como animal, pero se ha asociado
par-ticularmente a la carne del ganado bovino, ya que esta espe-cie
se ha considerado como un reservorio natural de este pa-tógeno. Sin
embargo existen reportes que mencionan su pre-sencia en otras
especies animales como cerdos, ovejas, caba-llos (Equs caballus),
venados, perros (Canis familiaris) y aves[2, 8, 16]. En relación
con la planta I, en su primera fecha demuestreo (20/02/2004) se
encontró que, si bien dos aisladosresultaron positivos para los
genes que codifican para los antí-genos O157 y H7, éstos no eran
portadores de ninguno de losdos genes que codifican para las
verotoxinas; sin embargo, enla segunda fecha de muestreo
(15/03/2004), de cuatro aisla-dos positivos para los genes rfbE y
fliC, tres fueron positivospara los genes de las verotoxinas 1 y 2
y el de la intimina. Esimportante mencionar que cada uno de estos
tres aislados fue-ron encontrados en cada uno de los tres
diferentes sitios demuestreo en la canal, es decir, uno en la
cadera, otro en elcostado y otro en el cuello, lo que plantea la
posibilidad que setratara de un mismo animal, o bien de animales
diferentespero procedentes de una misma granja o lugar y en la
cualexiste la presencia de E. coli O157:H7 enterohemorrágica.
141
_______________________________________________________________Revista
Científica, FCV-LUZ / Vol. XIX, Nº 2, 139 - 146, 2009
TABLA I
INICIADORES USADOS EN LAS REACCIONES DE PCR PARA LA
IDENTIFICACIÓN Y CARACTERIZACIÓN MOLECULARDE E. coli O157:H7 /
PRIMERS USED IN THE PCR REACTIONS FOR THE IDENTIFICATION AND
MOLECULAR CHARACTERIZATION
OF E. coli O157:H7
Gen Secuencia5´ � 3´
Tamaño (pb)
rfbE AAGATTGCGCTGAAGCCTTTGCATTGGCATCGTGTGGACAG
497
fliC GCGCTGTCGAGTTCTATCGAGCCAACGGTGACTTTATCGCCATTCC
625
stx1 CGCTGAATGTCATTCGCTCTGCCGTGGTATAGCTACTGTCACC
302
stx2 CTTCGGTATCCTATTCCC a
CTGCTGTGACAGTGACAAAACG b518
eaeA CAGGTCGTCGTGTCTGCTAAATCAGCGTGGTTGGATCAACCT
1087
a Se eliminaron dos guaninas en el extremo 3´. bSe eliminó una
citosina en el extremo 3´.
-
Respecto a los seis aislados que fueron encontrados enla planta
II y que amplificaron por PCR para los genes que co-difican para
los antígenos O157 y H7, cuatro fueron portadoresdel gen de la
verotoxina 1, y de estos sólo uno fue portador delgen de verotoxina
2 y el de intimina, y fue obtenido del cuellodel animal.
Si bien lo anterior señala el riesgo de encontrar a estepatógeno
en ambos tipos de canal y representa un riesgo parala salud, no
necesariamente implica que el animal sea el porta-dor, sino que
pone de manifiesto la ineficiencia de las buenasprácticas
higiénicas en el manejo de la canal, puesto que unanimal sano puede
portar el patógeno en su pelo, piel y tracto
142
Caracterización de aislados de E. coli O157:H7 en canales de
bovinos y porcinos mediante PCR / Gallegos, M. y
col.__________________
TABLA II
RESUMEN DE LA CARACTERIZACIÓN DE AISLADOS DE E. coli / SUMMARY
OF THE CHARACTERIZATION OF E. coli ISOLATES
Aislado Especie Planta Sitio de muestreoen la canal
Fecha demuestreo
Genotipo
rfbE fliC stx1 stx2 eaeA
1.1 Cerdo I B 20/02/2004 – – – – –
2.1 Cerdo I B 20/02/2004 – – – – –
3.1 Cerdo I C 20/02/2004 + + – – –
4.1 Cerdo I C 20/02/2004 + + – – –
5.1 Bovino II B 2/03/2004 + + + – –
6.1 Bovino II C 2/03/2004 + + + + +
7.1 Bovino II A 2/03/2004 + + + – –
8.1 Bovino II B 2/03/2004 + + + – –
10.1 Cerdo I A 15/03/2004 + + + + +
11.1 Cerdo I B 15/03/2004 + + + + +
12.1 Cerdo I C 15/03/2004 + + – – –
13.1 Cerdo I C 15/03/2004 + + + + +
14.1 Bovino II B 24/03/2004 + + – – –
15.1 Bovino II C 24/03/2004 + + – – –
A = falda. B = costado. C = cuello. + y – = respectivamente
portadores y no portadores del gen en cuestión.
497625
1 2 3 4 5 6 7 8 9 10 11 12 13 M
FIGURA 1. PRODUCTOS DE PCR A PARTIR DE LOS GENES fliC (625 pb) Y
rfbE (497 pb) EN AISLADOS DE E. coliOBTENIDOS DE CANALES DE CERDO Y
BOVINO. CARRILES 1-3 y 8-11 = AISLADOS A PARTIR DE CANALES DE
CERDO.CARRILES 4-7 = AISLADOS A PARTIR DE CANALES DE BOVINO. CARRIL
12 = E. coli O157:H7. CARRIL 13 = CONTROL
NEGATIVO. CARRIL M = MARCADOR DE PESO MOLECULAR HYPERLADDER 100
PB (BIOLINE)/ PCR PRODUCTS OF fliC (625pb) AND rfbE (497 pb) GENES
OF E. coli ISOLATES OBTAINED OF PORCINE AND BOVINE CARCASSES. LINES
1-3 AND 8-11 = ISOLATES
OBTAINED OF PORCINE CARCASSES. LINES 4-7 = ISOLATES OBTAINED OF
BOVINE CARCASSES. LINES 12 = E. coli O157:H7. LINE 13 =
NEGATIVE CONTROL. LINE M = HYPERLADDER MARKER 100 PB
(BIOLINE).
-
intestinal [2]; o bien la canal contaminada en el proceso de
sa-crificio contamine a otras canales debido a un deficiente
proce-so de evisceración. A pesar que el proceso de sacrificio
delganado bovino y porcino es diferente, las diferencias
observa-das entre el número de canales positivas de cerdo y
bovinopara cualquiera de los genes no fueron significativas
estadísti-camente (TABLA III), lo que sugiere la posibilidad de
encontrara E. coli O157:H7 enterohemorrágica indistintamente en
am-bos tipos de canal, sin embargo, estos datos deben conside-rarse
con reserva, dado el tamaño de muestras analizadas apesar que se
tiene concordancia con otros autores [4] quienesmencionan la
posibilidad que los cerdos sean hospederos bio-lógicamente
competentes para E. coli O157:H7 y otras cepasde E. coli
verotoxigénicas.
En algunos países como Estados Unidos de Norteaméri-ca, la
presencia del serotipo O157:H7 en los alimentos, no im-porta si es
o no verotoxigénica, es motivo de preocupación porel riesgo que
ello implica en la salud de los consumidores. Porlo tanto, además
de determinar la presencia de E. coliO157:H7 en los alimentos es
muy importante caracterizarlosen relación con otros factores de
patogenicidad. La importan-cia clínica que tiene E. coli O157:H7
como patógeno radica enel hecho que puede ser portadora de uno o
ambos genes quecodifican para las verotoxinas, así como el gen de
la intimina
lo que determina que la cepa sea considerada enterohemorrá-gica
[19]. La FIG. 2 muestra los productos de PCR a partir delgen stx1
de aislados que fueron positivos para los genes rfbEy fliC. Se
observa que no todos los aislados presentaron estegen, sólo siete
de ellos, correspondiendo cuatro a aislados ob-tenidos a partir de
canales de bovino y tres a partir de canalesde cerdo. Puesto que
los genes stx1 y stx2 se encuentrancada uno en un bacteriófago
temperado lisogénico, los cualesintegran su ADN al cromosoma de E.
coli O157:H7 [26], sepuede explicar porque algunas de las cepas de
E. coliO157:H7 expresaron sólo una o ambas verotoxinas, lo cual
yaha sido documentado [22].
En otro estudio [10] sobre la presencia de E. coliO157:H7 en
cerdos, se encontró además del genotipoO157:H7 portador de los
genes stx1, stx2 y eaeA, la presenciadel genotipo portador de stx1
y los genes de virulencia eaeA yhly, o bien, genotipos portadores
de eaeA, stx1 y stx2, pero nocon los cuatro genes stx1, stx2, eaeA
y hly. Así mismo otrosautores reportaron en canales de bovino, un
aislado de E. coliO157:H7 que fue negativa por PCR para los genes
stx peropositiva para los genes de virulencia ehx y eaeA [9]. De
losaislados que fueron caracterizados como portadores del genstx1,
cuatro fueron portadores del gen stx2, siendo tres de losaislados
obtenidos de canales de cerdo y uno de canal de bo-
143
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Científica, FCV-LUZ / Vol. XIX, Nº 2, 139 - 146, 2009
TABLA III
FRECUENCIA DE AISLADOS DE E. coli O157:H7 PORTADORES DE CADA UNO
DE LOS GENES rfbE, fliC, stx1, stx2, eaeA,Y DE LOS CINCO GENES POR
TIPO DE CANAL / FREQUENCY OF E. coli O157:H7 ISOLATES HARBORING
EACH OF THE GENES rfbE,
fliC, stx1, stx2, eaeA, AND THE FIVE GENES BY KIND OF
CARCASS
Tipo de canal rfbE fliC stx1 stx2 eaeA Cinco genes
Bovino 6/9 = 0,666 6/9 = 0,666 4/9 = 0,444 1/9 = 0,111 1/9 =
0,111 1/9 = 0,1111
Cerdo 6/9 = 0,666 6/9 = 0,666 3/9 = 0,333 3/9 = 0,333 3/9 =
0,333 3/9 = 0,3333
Significancia NS NS NS NS NS NS
NS = diferencia no significativa (P � 0,05).
1 2 3 4 5 6 M 7 8 9 10 11 12
302 pb
FIG. 2. PRODUCTOS DE PCR DEL sxt1 (302 PB) DE SUPUESTOS AISLADOS
DE E. coli O157:H7.CARRILES 1-2 Y 7-10 = AISLADOS A PARTIR DE
CANALES DE CERDOS. CARRILES 3-6 = AISLADOS A PARTIR
DE CANALES DE BOVINO. CARRIL 11 = E. coli O157:H7. CARRIL 12 =
CONTROL NEGATIVO. CARRIL M = MARCADORDE PESO MOLECULAR HYPERLADDER
100 PB (BIOLINE) / PCR PRODUCTS OF stx1 GENE (302 pb) OF PUTATIVE
E. coli O157:H7
ISOLATES. LINES 1-2 AND 8-11 = ISOLATES FROM PORCINE CARCASSES.
LINES 3-6 = ISOLATES FROM BOVINE CARCASSES. LINE 12 = E. coli
O157:H7. LINE 13 = NEGATIVE CONTROL. LINE M = HYPERLADDER MARKER
100 PB (BIOLINE).
-
vino (FIG. 3). No obstante se ha reportado una mayor propor-ción
de cepas portadoras del gen stx2 que del gen stx1 [9], enel
presente trabajo no se encontró la misma proporción, perosí hubo
coincidencia con otros reportes [16] donde mencionanque la mayoría
de las cepas portadoras del gen stx2, tambiénlo son para el gen
eaeA, ya que los cuatro aislados portadoresdel gen stx2 también
fueron portadores del gen eaeA (FIG. 4).
CONCLUSIONES
Las canales de bovino y las de cerdo pueden ser porta-doras de
E. coli O157:H7, lo que refleja la habilidad de estepatógeno para
colonizar también a la especie porcina. Los re-sultados obtenidos
en este trabajo a través de la PCR constitu-yen un aporte valioso
en materia de inocuidad alimentaria ysalud pública al demostrar la
presencia de E. coli O157:H7 en-terohemorrágica en estos tipos de
alimentos. De gran utilidadresultó la PCR para determinar el grado
de virulencia de losaislados ya que permitió identificar los que
fueron portadoresde los genes de virulencia verotoxinas 1 y 2 y del
gen de la in-timina, siendo además muy importante desde el punto de
vistaepidemiológico el hecho de haber encontrado cepas portado-ras
de los factores de virulencia por ser consideradas patóge-nas al
humano. Debido a que no se obtuvieron muestras am-bientales, ni de
los trabajadores dentro de los mataderos, asícomo de las
herramientas de corte que ellos utilizan, serán ne-cesarios
estudios posteriores para descartar la existencia defuentes
externas de contaminación, así como la posibilidad decontaminación
cruzada al momento de la evisceración. Deacuerdo con los resultados
encontrados, se abre la posibilidadpara realizar estudios más a
fondo del destino que tendrían eltipo de cepas de E. coli O157:H7
sobre la incidencia de enfer-medades transmitidas por los
alimentos, o si bien, el procesode cocción es suficiente para
eliminar el riesgo de contamina-ción al momento del consumo de la
carne contaminada con di-cha bacteria.
144
Caracterización de aislados de E. coli O157:H7 en canales de
bovinos y porcinos mediante PCR / Gallegos, M. y
col.__________________
1 2 3 4 5 M 6 7 8 9
518 pb
FIG. 3. PRODUCTOS DE PCR DEL GEN sxt2 (518 PB) DE SUPUESTO
AISLADOS DE E. coli O157:H7.CARRILES 1-4 = AISLADOS A PARTIR DE
CANALES DE BOVINO. CARRILES 5 Y 6-7 = AISLADOS A PARTIR DE
CANALES
DE CERDO. CARRIL 8 = E. coli O157:H7. CARRIL 9 = CONTROL
NEGATIVO. CARRIL M = MARCADOR DE PESOMOLECULAR HYPERLADDER 100 PB
(BIOLINE) / PCR PRODUCTS OF stx2 GENE (518 PB) OF PUTATIVE E. coli
O157:H7 ISOLATES.
LINES 1-4 = ISOLATES FROM BOVINE CARCASSES. LINES 5 AND 7-8 =
ISOLATES FROM PORK CARCASSES. LINE 9 = E. coli O157:H7. LINE 10
=
NEGATIVE CONTROL. LINE M = HYPERLADDER MOLECULAR MARKER 100 PB
(BIOLINE).
M 1 2 3 4 5 6
1087 pb
FIG. 4. PRODUCTOS DE PCR DEL GEN eaeA (1087 PB) DESUPUESTOS
AISLADOS DE E. coli O157:H7. CARRIL M =MARCADOR DE PESO MOLECULAR
HYPERLADDER 100PB (BIOLINE). CARRIL 1 = AISLADO A PARTIR DE CANALDE
BOVINO. CARRILES 2-4 = AISLADOS A PARTIR DECANALES DE CERDO. CARRIL
5 = E. coli O157:H7. CA-RRIL 6 = CONTROL NEGATIVO / PCR PRODUCTS OF
eaeAGENE (1087 PB) OF PUTATIVE E. coli O157:H7 ISOLATES. LINE M
=
HYPERLADDER MOLECULAR MARKER 100 PB (BIOLINE). LINE 2 =
ISOLATE FORM BOVINE CARCASS. LINES 3-5 = ISOLATES FROM
PORK CARCASSES. LINE 6 = E. coli O157:H7. LINE 7 = NEGATIVE
CONTROL.
-
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bovinos y porcinos mediante PCR / Gallegos, M. y
col.__________________
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M:Fo
odMi
crob
iolog
y&
Safet
y
JFS M: Food Microbiology and Safety
PCR Detection and Microbiological Isolation ofSalmonella spp.
from Fresh Beef and CantaloupesM.A. GALLEGOS-ROBLES, A.
MORALES-LOREDO, G. ÁLVAREZ-OJEDA, J.A. OSUNA-GARCÍA, I.O.
MART́INEZ,L.H. MORALES-RAMOS, AND P. FRATAMICO
ABSTRACT: Species belonging to the genus Salmonella are an
important cause of enteric fevers, gastroenteritis,and septicemia,
and the pathogens are commonly transmitted through contaminated
food. In this study, poly-merase chain reaction (PCR) amplification
of a 287-bp region of the invA gene was compared to a
microbiologicaltechnique to determine the presence of Salmonella in
retail beef and in cantaloupe rinse samples. Both methodsshowed the
same level of sensitivity, detecting 1 CFU/25 g of meat after
enrichment for 24 h at 42 ◦C. The presenceof Salmonella was
determined in 50 commercial top sirloin beef samples that were not
artificially inoculated. Threesamples were positive by the
microbiological method, and these samples and an additional sample
were positive bythe PCR. Both methods were also used to test
surface rinses of cantaloupes collected from 4 farms in Nayarit,
Mex-ico. Salmonella was detected by the microbiological method in 9
of 20 samples (45%), whereas the pathogen wasdetected by the PCR in
11 samples (55%). This study demonstrates the utility of the PCR
targeting the invA gene todetermine the presence of Salmonella spp.
in beef and cantaloupe samples.
Keywords: cantaloupe, detection, meat, PCR, Salmonella
Introduction
Salmonellosis caused by species in the genus Salmonella
wasdescribed in 1984 as a “new and significant threat to the
pub-lic health” by the World Health Organization (FAO 1984),
andSalmonella has remained a major foodborne pathogen
associatedwith different types of food. Gutiérrez and others
(2000) reportedthe isolation of Salmonella spp. in Mexico in 51% of
fast foodsamples, 23% of processed meat products (ham, chorizo, and
ba-con), 22% of ground food samples (beef, chicken, fish), 3% of
milkproducts, and in 1% of both fresh and powdered eggs.
Salmonel-losis outbreaks in the United States linked to the
consumption ofcantaloupes implicated Salmonella serotypes Saphra
and Poonaas the causative agents from cantaloupes that originated
fromMexico (Mohle and others 1999; CDC 2002). Analyses of fruits
andvegetables imported to the United States in 1999 showed that
of1003 analyzed samples, 35 (3.5%) tested positive for
Salmonellaspp., and of these, 8 were cantaloupe samples (22.9%),
indicatingthat cantaloupe was the 2nd most contaminated type of
productafter cilantro (FDA 2001). Currently in Mexico, the official
proce-dure for detection of Salmonella spp. is a cultural method,
and thisprocedure could take from 3 to 5 d for confirmation, which
is adisadvantage when the results are needed promptly (SSA 1994;
Pe-plow and others 1999). Molecular methods, such as the
polymerase
MS 20080484 Submitted 6/29/2008, Accepted 10/1/2008. Author
Gallegos-Robles is with Facultad de Agricultura y Zootecnia, Univ.
Juárez del Es-tado de Durango, Venecia, Gomez Palacio, Durango,
México, C.P. 35000.Author Morales-Loredo is with Consorcio
Técnico del Noreste de México,A.C. UGRNL. Km. 4.5, Carretera a
Reynosa. Guadalupe, N.L. México., C.P.67100. Authors
Álvarez-Ojeda and Osuna-Garcı́a are with Inst. Nacionalde
Investigaciones, Forestales, Agrı́colas y Pecuarias (INIFAP),
Guadalupe,Nuevo León, México, C.P. 67100. Authors Martı́nez and
Morales-Ramosare with Facultad de Ciencias Biológicas, Univ.
Autónoma de NuevoLeón, San Nicolás de los Garza, Nuevo León,
México. Author Fratam-ico is with USDA, Agricultural Research
Service, Eastern Regional Re-search Center, Microbial Food Safety
Research Unit, 600 E. Mermaid Lane,Wyndmoor, PA 19038 U.S.A. Direct
inquiries to author Morales-Loredo(E-mail:
[email protected]).
chain reaction (PCR), have shown high sensitivity and
specificity fordetecting target pathogens, including Salmonella, in
different typesof foods, and the time required to obtain results
can be as short as12 h (Ferretti and others 2001; Croci and others
2004). However, mi-crobiological techniques are used as reference
methods to demon-strate the efficacy and validity of new techniques
(Fernandez 2000).The objective of this study was to compare the
sensitivity of a PCRassay to a microbiological method and to
evaluate the 2 methodsfor the detection of Salmonella spp. in
naturally contaminated beefand cantaloupe rinse samples.
Materials and Methods
Bacterial strain, growth conditions, and preparationof
inoculum
S. Typhimurium ATCC 13311 was grown on trypticase soy agar(TSA)
(Becton Dickinson Co., Sparks, Md., U.S.A.) at 37 ◦C for 24
h.Afterward, 10 mL of physiological saline solution (0.85%)
wereadded to the plate to obtain a homogeneous suspension of
bacte-ria. An aliquot of the suspension was diluted to a
concentration ofthe nr 5 tube of the MacFarland scale (1.5 × 108
CFU/mL). Ten-foldserial dilutions were prepared in saline solution
to give suspensionscontaining 100 to 104 CFU/mL.
Artificial inoculation of top sirloin meat samplesThe top
sirloin meat samples were obtained on the same date
from supermarkets in Monterrey, Nuevo León, Mexico. Meat
sam-ples were placed into a cooler (at 4 ◦C) and transported to
thelaboratory. The samples were immediately separately
inoculatedwith the previously mentioned dilutions as described
subsequently.Three sterile plastic bags (Whirl-Pak
R©, Nasco, Modesto, Calif.,
U.S.A.) with the capacity of 500 mL were labeled for each
dilu-tion, and 25 g of top sirloin meat were weighed and placed
ineach plastic bag with 225 mL sterile buffered peptone water
(BPW)(Becton Dickinson and Co.). An uninoculated control sample
was
C© 2008 Institute of Food Technologists R© Vol. 74, Nr. 1,
2009—JOURNAL OF FOOD SCIENCE M37doi:
10.1111/j.1750-3841.2008.01006.xFurther reproduction without
permission is prohibited
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Detection of Salmonella spp. in food . . .
included to ensure that the meat was not naturally
contaminatedwith Salmonella. One milliliter of each bacterial
dilution (100 to 104
CFU/mL) was added to the corresponding plastic bag, which
wasthen mixed by hand for 2 min followed by incubation at 35 ◦C
for24 h. The enrichment and microbiological analyses were
performedusing tetrathionate (TT) broth (Becton Dickinson Co.)
according tothe method described in the Microbiology Laboratory
Guidebook(FSIS-USDA 2004). The experiments were performed in
triplicate.
DNA extraction from inoculated top sirloin meatsamples
Three milliliters from the Tetrathionate broth enrichments
wereused to form cell pellets by centrifuging at 3000 rpm for 5
min.DNA extraction from the cell pellets was performed using the
CTAB(cetyl trimethyl ammonium bromide) method; however, the use
ofpolyvinylpyrrolidone and ß-mercaptoethanol was omitted (Doyleand
Doyle 1987). The extracted DNA was stored at −20 ◦C.
PCR amplification conditionsAmplification of the target sequence
was performed using a PCR
Express thermal cycler (PCR Express; Thermo Hybaid,
Middlesex,U.K.). The PCR mixture contained 25 pmoles of each of the
primerstargeting the invA gene (Rahn and others 1992), 200 μM of
eachof the 4 deoxynucleoside triphosphates (Bioline Inc.,
Randolph,Mass., U.S.A.), 1 mM MgCl2, 1× Reaction Buffer (200 mM
Tris-HClpH 8, 500 mM KCl), 2.5 U of Taq DNA polymerase (Promega,
Madi-son, Wis., U.S.A.), 100 ng of DNA template, and deionized
water fora final volume of 25 μL. The reaction mixture was
subjected to thefollowing thermal cycling conditions: heat
denaturation at 95 ◦Cfor 1 min, and then 35 cycles with heat
denaturation at 95 ◦C for30 s, primer annealing at 58 ◦C for 30 s,
and DNA extension at72 ◦C for 30 s. After the last cycle, samples
were maintained at 72 ◦Cfor 10 min to complete synthesis of all
strands. The PCR productswere subjected to gel electrophoresis
(1.5% agarose; Promega), andthen stained with ethidium bromide (0.5
μg/mL), visualized with aUV transilluminator (Spectroline
Transilluminator, Model 7C-254R.Electronics Corp., Westbury, New
York, U.S.A.), and photographed.
Detection of Salmonella spp. in samples ofcommercial bovine
meat
Fifty different samples of bovine meat (New York Strip andRib
Eye) obtained from different supermarkets located in
themetropolitan area of Monterrey, N. L. Mexico, were collected
dur-ing the month of May in 2005 to determine if the meat was
natu-rally contaminated with Salmonella spp. Handling of the
samplesand the microbiological and PCR methods were performed as
de-scribed previously.
Detection of Salmonella spp. from rinses ofcantaloupe
surfaces
Cantaloupe melons were obtained at 4 fields in Nayarit,
Mexico,on 4 different dates from February 25 to April 11 of 2005 (1
field perdate). Each field was divided into 5 representative
quadrants (I toV). Twenty-five melons were collected from each
field, 5 melons perquadrant. The melons were randomly collected,
and adhered par-ticles of soil were not removed. Melon surface
washings were doneat the field. Each melon was placed in a sterile
Whirl-Pak bag usinggloves that were changed with each melon.
Twenty-five milliliters of0.1% sterile buffered peptone water were
added to each plastic bag,and the fruit was washed thoroughly by
shaking and mixing the bagby hand for at least 2 min. The washings
from the 5 melons ob-tained from the same quadrant were combined
into a single sterileglass bottle, and then placed into a cooler
(at 4 ◦C) and transported
to the laboratory. Processing of the samples was performed
within24 h of obtaining the rinses. At the laboratory, each sample
wasmixed by shaking, and 25 mL were removed and added to 225 mLof
BPW to continue with the microbiological method and the PCRassay as
described previously.
Results and Discussion
Isolation of Salmonella spp. from artificiallyinoculated meat
samples and detection by the PCR
The PCR assay and the microbiological method showed an
equallevel of sensitivity with a limit of detection for S.
Typhimuriumby both methods of 1 CFU/25 g of meat (initial inoculum
level)(data not shown). The primer pair targeting the invA gene,
de-signed by Rahn and others (1992), was tested using a collection
of630 Salmonella strains and showed 99.4% specificity and no
am-plification of DNA from non-Salmonella strains. Moreover, the
ad-equacy of invA as a target gene in PCR assays was tested by
Daumand others (2002) in a fluorogenic TaqMan PCR assay to
confirmthe presence of Salmonella directly from chicken in less
than 3 h.Although in the current study a single serovar was tested
using thisassay, we have also amplified the PCR product of the invA
gene inserovar S. Enteritidis using the same primer pair in assays
for thedetection of Salmonella in other types of food samples
(unpub-lished data). Thus, the PCR assay targeting the invA gene
can po-tentially be used to detect Salmonella in raw beef samples
as analternative to the cultural method. However, enrichment is a
nec-essary step if fresh products are tested by either the
microbiolog-ical method or the PCR, since it is possible that a low
level of thepathogen of interest may be present. Growth of the
target organ-ism, however, could be inhibited somewhat by the
accompanyingflora, which might give a negative result by the
cultural method, orthe sensitivity of the PCR assay could be
decreased. As such, it is im-portant to select a suitable
enrichment medium to inhibit the back-ground flora and the
appropriate enrichment time because PCRsensitivity tends to
increase with increases in enrichment times(Guo and others
2000).
Microbiological assay and PCR of commercial meatsamples
The data in Table 1 show results of 4 samples, which were
pos-itive by the PCR assay and the results of the same samples
testedusing the cultural method for the detection of Salmonella
spp. Theother 46 meat samples analyzed were negative using both
meth-ods. Three samples out of 50 (6%) were detected as positives
bythe microbiological method, while the PCR assay detected an
ad-ditional sample (8%). Several reasons could explain why a
samplewas detected as positive by the PCR (sample nr 12) and not
de-tected as positive by the microbiological method. It is possible
thatthe cells in the meat sample were injured due to the use of
sani-tation products or due to stressful storage conditions, which
candamage or even kill the Salmonella. This in turn would affect
theability to detect the pathogen using the cultural method, since
thismethod is dependent on growth of the cells. Figure 1 shows
the
Table 1 --- Meat samples positive for Salmonella spp., byboth
the cultural and PCR methods.
Sample nr Cultural method PCR assay
3 + +12 − +33 + +41 + +
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PCR products obtained following amplification of the positive
meatsamples. Sample nr 12 (lane 3) shows a weak band. Thus, it is
pos-sible that there was less PCR product from this sample
becausethere were a lower number of cells after enrichment compared
tothe other samples. This was likely the reason why the pathogen
wasnot detected by plating. Although, the sensitivity of both the
mi-crobiological and PCR methods was the same (1 CFU/25 g) whenmeat
samples were inoculated with nonstressed cells, in
naturallycontaminated samples, the cells may be stressed affecting
theirability to grow as rapidly as nonstressed cells in the
enrichmentmedium.
Cultural and PCR assays using cantaloupe rinsesamples
The data in Table 2 show that in the 1st field, 4 positive
sam-ples were detected (quadrants I to III and V) by the
microbiologicalmethod, and there were 4 positives from the same 4
quadrants bythe PCR. However, for quadrants III and IV, the results
of both meth-ods differed. In the 2nd field, the results of both
methods were thesame, with positive results from samples in all 5
quadrants. In the3rd and 4th fields, all results using the
microbiological assay werenegative, while using the PCR, results
were positive from samplesfrom the 3rd field from quadrants II and
III and were negative fromthe 4th field from all 5 quadrants. With
the exception of samplesfrom quadrant III and IV from the 1st field
where results differed,results of the PCR assay agreed with those
of the cultural method,and in some cases the PCR assay was more
sensitive for detection ofSalmonella (quadrants II and III of the
3rd sampling). In summary,Salmonella spp. were detected by the
microbiological method in 9of 20 samples (45%), whereas the
pathogen was detected by the PCRin 11 samples (55%). These results
are partially in agreement withthose of Espinoza-Medina and others
(2006) who found that bythe PCR method, 25.7% of samples from
in-field cantaloupes were
Figure 1 --- PCR products from the Salmonella invA gene inbeef
samples positive for Salmonella spp. Lane 1, molecu-lar weight
markers, 100-bp ladder (BIOLINE); lanes 2 to 5,samples 3, 12, 33,
and 41, respectively; lane 6, negativecontrol; and lane 7, positive
control --- S. Typhimurium.
Table 2 --- Positive results for Salmonella spp. from cantaloupe
surface washings using the cultural method and thePCR assay.
Assay results
First field Second field Third field Fourth field
Quadrant Microbiological PCR Microbio logical PCR
Microbiological PCR Microbiological PCR
I + + + + − − − −II + + + + − + − −III + − + + − + − −IV − + + +
− − − −V + + + + − − − −
positive for Salmonella, whereas no positive samples were
detectedby the standard method.
The detection of this pathogen by the PCR was done from an
en-richment culture; therefore, in addition to growth of
Salmonella,the microflora from the melon samples also grew. Thus,
the PCRwas sensitive and specific, since Salmonella was detected in
thepresence of other microorganisms found in the melon produc-tion
environment. Previous microbiological studies conducted inthe
Lagunera Region of Mexico (Froto and others 2004) found thepresence
of bacteria that belonged to the Enterobacteriaceae fam-ily and
other microorganisms in the cuticle of melons, as well asplant
pathogens such as Fusarium spp., Verticillium spp., and
Rhi-zoctonia solani, and saprophytes such as Aspergillus spp.,
Rhizo-pus spp., and Penicillium spp. Also, human pathogens,
includingClostridium botulinum, Listeria monocytogenes, Vibrio
cholerae,Brucella melitensis, Salmonella Typhi, Salmonella
Paratyphi, hep-atitis A virus, Escherichia coli, and Shigella
dysenteriae, were found.Most of these pathogens could be found in
the soil coming frombovine and avian manure and from human feces
(Froto andothers 2004). The absence of the Salmonella invA sequence
in otherinvasive bacteria such as Yersinia spp., Shigella spp., and
enteroin-vasive E. coli, which also have the capacity to invade
epithelial cells,demonstrates the particular specificity and
utility of this primerpair for detection of Salmonella spp. (Galán
and Curtiss 1991). TheinvA gene has been used as the target in PCR
assays mainly for de-tecting Salmonella in poultry, meats, and
dairy products, and invegetables and fruits (Guo and others 2000).
The PCR results of thecurrent study shown in Figure 2 indicate that
the primers could beused for detection of Salmonella from
cantaloupe surface washingsand potentially in other types of fruits
and vegetable samples, aswell.
The differences in results from field to field may have been
dueto differences in levels of Salmonella contamination. This may
havebeen influenced in part by changes in the environment,
includingloss of specific nutrients and fluctuations in humidity,
temperature,and ultraviolet light, all of which could damage
bacterial cells and
Figure 2 --- Detection of Salmonella spp., by the PCR
fromcantaloupe surface washings. Lanes 1 to 4, quadrants Ito IV
(3rd field); lanes 5 to 6 and 8 to 10, quadrants I to V(4th field);
lane 7, molecular weight marker, 100-bp lad-der (BIOLINE); lane 11,
positive control --- S. Typhimurium;lane 12, negative control.
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Detection of Salmonella spp. in food . . .
combined with the low probability that human pathogens
developstress resistance (Dickinson 1986; O’Brien and Lindow 1988).
Fur-thermore, each field was sampled only once during the 1.5
moperiod, and Good Agricultural Practices (GAP) were not appliedin
the first 3 fields. For the 4th field, in which no positive
resultswere obtained, the cantaloupes were obtained from a field in
whichGAP were applied. These included the use of plastic mulch,
fertir-rigation, water without evidence of microbiological
contamination,portable restrooms in the fields, training of field
workers in GAP, useof authorized pesticides, designation of areas
where workers mayeat and take breaks, and availability of potable
water for workers,which according to HACCP (Hazard Analysis and
Critical ControlPoint) plans are basic tools for reducing physical,
chemical, and mi-crobiological hazards in agricultural
production.
Conclusions
Although meat is usually not consumed raw, there is risk
ofSalmonella infection if the meat is improperly cooked, andthere
is also the possibility of cross contamination of Salmonellawith
foods that are consumed raw. Produce may become con-taminated in
the field through the use of contaminated irrigationwater or manure
or also from animals or inadequate worker hy-giene. Therefore, the
ability to rapidly detect Salmonella in meat,fruit, and other foods
could lower the risk of contaminated foodreaching the consumer. Use
of a sensitive assay for detection ofSalmonella in melons is also
very important, since this food is eatenraw. The PCR assay
evaluated in the current study could be used asa screening test,
since results would be available in less time thanwith the cultural
method. PCR-positive results could then be con-firmed by the
cultural method. Because the invA gene is presentin pathogenic
Salmonella serotypes, the PCR assay based on theprimer pair
targeting this gene could be applied for detection ofSalmonella
spp. that may be associated with particular food prod-ucts,
including poultry and food products that are consumed rawsuch as
fruits and vegetables and/or ready-to-eat food. Further re-search
will focus on validating the robustness of the SalmonellaPCR assay
in approved laboratories in Mexico for its use as a screen-ing test
using different types of samples, along with confirmationof the
pathogen by the microbiological method. Additional stud-ies to
determine the most prevalent Salmonella serotypes found inbeef and
in cantaloupes and to determine the effect of environmen-tal
changes and the use of GAP on the prevalence of Salmonella inthese
foods are warranted.
AcknowledgmentsThe Consejo Nacional de Ciencia y Tecnologı́a is
acknowledged forfunding this research.
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