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Applied and Environmental Microbiology, December 2000, p. 5273-5281, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Suitability of PCR Fingerprinting,
Infrequent-Restriction-Site PCR, and Pulsed-Field Gel
Electrophoresis, Combined with Computerized Gel Analysis, in
Library Typing of Salmonella enterica Serovar
Enteritidis
Javier
Garaizar,1,*
Nuria
López-Molina,1
Idoia
Laconcha,1
Dorte
Lau
Baggesen,2
Aitor
Rementeria,1
Ana
Vivanco,1
Ana
Audicana,3 and
Ildefonso
Perales3
Department of Immunology, Microbiology and
Parasitology, Basque Country University, 01080 Vitoria-Gasteiz,1 and Laboratory of
Microbiology, Public Health Laboratory, Basque Government, 48010 Bilbao,3 Spain, and Danish
Veterinary Laboratory, Copenhagen V, Denmark2
Received 25 May 2000/Accepted 3 October 2000
 |
ABSTRACT |
Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a
library typing computerized system for serovar Enteritidis on the basis
of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or
pulsed-field gel electrophoresis (PFGE). The rate of PCR fingerprinting
interassay and intercenter reproducibility was low and was only
increased when DNA samples were extracted at the same time and
amplified with the same reaction mixtures. Reproducibility of IRS-PCR
technique reached 100%, but discrimination was low (D = 0.52). The PFGE procedure showed an intercenter reproducibility value
of 93.3%. The high reproducibility of PFGE combined with the
previously determined high discrimination directed its use for library
typing. The use of PFGE with enzymes XbaI,
BlnI, and SpeI for library typing of serovar
Enteritidis was assessed with GelCompar 4.0 software. Three computer
libraries of PFGE DNA profiles were constructed, and their ability to
recognize new DNA profiles was analyzed. The results obtained pointed
out that the combination of PFGE with computerized analysis could be
suitable in long-term epidemiological comparison and surveillance of
Salmonella serovar Enteritidis, specially if the prevalence
of genetic events that could be responsible for changes in PFGE
profiles in this serovar was low.
| |
INTRODUCTION |
Salmonella enterica
serovar Enteritidis is widely recognized as a major cause of food-borne
gastroenteritis in humans and has been isolated from cases of human
disease in increasing numbers worldwide during the past 20 years
(25). Animals and their products, particularly meat and eggs
from chickens, are considered major sources of infections with this
pathogen for humans (19, 22). Phage typing (PT) has
facilitated epidemiological tracing, and it has become clear that most
isolates of serovar Enteritidis belong to a limited number of PTs
(35). It has been suggested by many authors that PT data
reveal the clonal diffusion of a limited number of strains throughout
Europe and the United States (17, 25).
During the past 15 years, an expansion of techniques suited for DNA
amplification has been observed. PCR procedures have confirmed their
enormous potential for the diagnosis of infectious disease agents.
Besides, Williams et al. (37) and Welsh and McClelland (36) developed a general PCR typing procedure, based on the use of a short single arbitrary oligonucleotide and low annealing temperatures. These variations in conventional PCR procedure allowed for the generation of genotypic markers, and validation at taxonomic and epidemiological levels has been achieved for a number of
microorganisms (31). We have demonstrated (13)
the usefulness of combining PCR fingerprinting with PT in the
epidemiological characterization of Salmonella serovar Enteritidis.
Infrequent-restriction-site PCR (IRS-PCR) is a recently developed
typing method, based on the selective amplification of DNA fragments
generated by the double restriction of DNA. The method is carried out
in four steps: DNA digestion with two restriction enzymes, DNA
ligation of oligonucleotide adapters to the cohesive ends of the
restricted fragments, selective amplification by PCR, and separation of
PCR-generated fragments by gel electrophoresis. The method has been
shown to be easy to perform and to have good reproducibility (15,
24). Results of the application of this new technology, in
relation with the epidemiological typing of Salmonella
serovar Enteritidis, have not been reported in the literature yet.
Pulsed-field gel electrophoresis (PFGE) is an established method for
the analysis of large fragments generated by restriction endonuclease digestion of genomic DNA (14) and is
currently considered to be one of the most reliable typing procedures
(16). The PFGE method has been shown to be highly
effective for epidemiological studies of some serovars of
S. enterica (2, 3, 17, 20, 21). The discrete
number of well-resolved band fragments allows a visual comparison
of the restriction profiles. Furthermore, the adequate typeability and
discriminatory power of this method have led to the conclusion that
PFGE is, at present, one of the most valuable epidemiological tools
available for the molecular analysis of this important pathogen
(10, 11, 23). However, to further evaluate the potential of
this molecular typing system, it must be demonstrated to be
sufficiently reproducible or amenable to standardization for use in
definitive library typing (33).
For the establishment of an international database of
Salmonella DNA profiles, it is necessary to build up large
databases containing fragment patterns from a wide variety of strains,
to which unknown strains can be compared. Computerized gel analysis allows the comparison of band profiles presented in different gels and
the construction of databases that could be useful in the
development of library typing systems of microorganisms
(28). Band profiles are compared and clustered in
dendrograms on the basis of calculated similarity coefficients.
The objective of this study was to explore the possibilities of
establishing such a library typing system for
Salmonella serovar Enteritidis on the basis of PCR
fingerprinting, IRS-PCR, or PFGE. The study was designed to determine
the intralaboratory or intercenter reproducibility of the methods in
three different laboratories, comparing the DNA patterns obtained with
an established protocol. Computerized gel analysis was used for the
construction of DNA profile databases, for assessment of the
similarities between band profiles, and for the detection and
identification of new strains through their DNA profiles.
| |
MATERIALS AND METHODS |
Bacteria.
Strains of S. enterica (n = 212) were divided into five bacterial collections and were used
for several purposes (Table 1). The
isolates were identified by conventional biochemical methods and
serotyped with respect to cell wall (O) and flagellar (H) antigens. The
isolates belonging to serovar Enteritidis were phage typed by standard
methods and assigned to phage types according to the scheme of Ward et
al. (35).
PCR fingerprinting.
Fifty isolates of Salmonella
serovar Enteritidis, isolated from humans, foods, and environmental
sources at the Public Health Laboratory, Bilbao, Spain, were included
in the study of reproducibility of PCR fingerprinting (Table 1,
collection 1). Twenty-seven of the isolates were derived from eight
outbreaks of human salmonellosis in the Basque Country, Spain, in the
period between 1983 and 1994. The rest of the isolates were
epidemiologically unrelated strains recovered from food and water
reservoirs, rivers, and beaches in the same region. The type strain of
Salmonella serovar Enteritidis ATCC 49214 from the American
Type Culture Collection was also included. The strains were analyzed
separately in two laboratories: Laboratory of Microbiology, Public
Health Laboratory, Bilbao, Spain (center A), and Department of
Immunology, Microbiology, and Parasitology, Basque Country University,
Vitoria-Gasteiz, Spain (center B). The isolates were grown overnight at
37°C in tryptone soy agar (BBL, Cockeysville, Md.). A small loopful
of bacteria was used for the DNA extractions. After being heated at
100°C for 10 min and centrifuged in a Micro Centaur centrifuge (MSE,
Sanyo, Leicester, United Kingdom) (13,000 rpm, 10 min), the DNA
concentration of the supernatant was measured by spectrophotometry at
an absorbance of 260 nm. A stock solution of 50 ng of DNA per µl was
kept at 
20°C. The following three individual primers were used in
this study (Table 2): ERIC2, M13, and
OPS-19 (synthetized by PE Applied Biosystems, Madrid, Spain). The
amplification temperatures and the controls used were as described
previously by López-Molina et al. (13). In order to
optimize the PCR procedures, variable amounts per reaction of the
following reagents were evaluated: primer (10 to 90 pmol),
MgCl2 (0 to 4 mM), and DNA template (100 to 325 ng).
Finally, PCR was carried out in a 50-µl volume with 10 mM Tris HCl
(pH 8.3), 50 mM KCl, 3 mM MgCl2, 200 µM each
deoxynucleoside triphosphate (PE Applied Biosystems), 1 U of
Ampli-Taq polymerase (PE Applied Biosystems), 250 ng of
template DNA, and primer (50 pmol for ERIC2, 40 pmol for M13, and 20 pmol for OPS-19). Two brands of thermocyclers were used in this study.
A 9600 thermocycler (PE Applied Biosystems) was located in center A and
an Autocycler 32 thermocycler (Linus, Mortlake, Australia) was used in
center B. After amplification, DNA was subjected to horizontal
electrophoresis on 2% agarose (Bio-Rad Laboratories, Richmond, Calif.)
in Tris-borate-EDTA (TBE) buffer for 1 h and 30 min at 150 V. pGEM
(Promega, Barcelona, Spain) was used as a molecular weight standard and
was allocated along the gels several times in order to normalize the
gels in the computerized analysis.
IRS-PCR.
A collection of nine strains belonging to different
Salmonella serovars recovered from human and environmental
sources at the Public Health Laboratory, Bilbao, Spain, was used to
optimize the IRS-PCR method (Table 1, collection 2). A bacterial
collection of 36 strains of Salmonella serovar Enteritidis
was used to study the typability, the discriminatory power, and the
reproducibility of the IRS-PCR method (Table 1, collection 3). The
strains were recovered from unrelated animal, human, or food sources
and identified as described above. Ten strains were isolated in Denmark
between 1985 and 1997 and were provided by the Danish Veterinary
Laboratory (Copenhagen, Denmark) (center C). S. Chappell, from the
Central Veterinary Laboratory, Surrey, United Kingdom, kindly provided 13 strains isolated in 1996 and 1997. The remaining 13 strains were
isolated between 1984 and 1992 at the Public Health Laboratory, Bilbao,
Spain. The IRS-PCR study was performed at center B.
Two DNA extraction procedures were used and compared. The first
procedure was as described above in the PCR fingerprinting
method. A
second procedure was as described previously by Garaizar
et al.
(
5). The DNA concentration was estimated as described
in
"PCR fingerprinting," and stock solutions of 0.4 µg of DNA
per
µl were kept at
20°C. The adapters and primers used in IRS-PCR
are shown in Table
2, and the IRS-PCR method was performed according
to
the protocol previously described by Mazurek et al. (
15).
Two combinations of restriction enzymes (Boehringer-Mannheim,
Barcelona, Spain) were evaluated:
XbaI-
HhaI and
XbaI-
TaqI. DNA
restriction was carried out with 1 µg of bacterial DNA, 10 U of
XbaI, and 10 U of
HhaI (or the same amount of
TaqI) and was
performed
for 1 h at 37°C. The following reagents were added to
the reaction
mixture: 2 U of T4 DNA ligase (Bioline, London, United
Kingdom),
1 mM ATP (Bioline), 20 pmol of adapter AX (AX1-AX2), and 20 pmol
of adapter AH (AH1-AH2) (or the same amount of adaptor AT
[AT1-AT2]
when the
XbaI-
TaqI combination was
used). All the oligonucleotide
adapters and primers were synthesized by
PE Applied Biosystems.
The DNA ligation was carried out at 16°C for
1 h. In order to
inactivate the T4 ligase, the samples were heated
at 65°C for
20 min. A second round of DNA restriction was carried out
with
5 U of each restriction enzyme for 15 min at 37°C. The PCR was
carried out in a final volume of 50 µl containing the following
reagents: 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl
2,
200
µM each deoxynucleoside triphosphate, 1 µM primer PX (Table
2),
1 µM primer AH1 (or 1 µM primer AT1 when the
XbaI-
TaqI combination
was used), 1 U of
Ampli-
Taq polymerase, and 1 µl of the restricted
ligated
DNA. An Autocycler 32 thermocycler (Linus) was used with
the following
amplification temperatures: 1 cycle at 94°C for
6 min and 30 cycles
at 94°C for 1 min, 60°C for 1 min, and 72°C
for 2 min. The
amplified DNA fragments were electrophoresed in
8% polyacrylamide gel
in TBE buffer at a constant 150 V for 45
min. pGEM was used as the
molecular weight
standard.
PFGE.
The bacterial collection used to study the intercenter
reproducibility of PFGE was composed of 15 unrelated
Salmonella serovar Enteritidis strains recovered from human,
food, and animal sources, isolated in Denmark, England, and Spain
between 1984 and 1997 (Table 1, collection 4). The set of strains was
analyzed separately in two laboratories, center B and center C. In both
centers, DNA preparations and fragmentation with restriction
endonucleases XbaI, BlnI, and SpeI
were performed according to the protocol previously described by Olsen
et al. (17). Each laboratory prepared the required solutions
and used different batches of commercial restriction endonucleases
(Boehringer-Mannheim and Amersham Life Science, Ltd., Buckinghamshire,
England, respectively). Samples were subjected to PFGE on 1.2% agarose
(wt/vol) in 0.5× TBE buffer. The running conditions for different
enzymes were as described by Laconcha et al. (11). The
electrophoresis apparatus used was a CHEF-DR III (Bio-Rad) in center C
and a CHEF-DR II in center B. Lambda DNA (Bio-Rad and Sigma, St. Louis,
Mo.) served as a molecular size standard.
Computerized gel analysis.
After electrophoresis, the gels
with the DNA were stained with ethidium bromide and photographed with
Polaroid film. Photographs of PFGE fingerprints from the different gels
were interpreted visually according to published guidelines (27,
29). A capital letter was used to define a distinct type, and
subtype profiles were indicated by a numerical suffix. The index of
discrimination was calculated according to Simpson's index
(D) of diversity as described by Hunter and Gaston
(9). For computer analysis, images were scanned, saved in a
TIFF format as described by Garaizar et al. (6), and sent
intercenter by file transfer protocol, and then analyzed by GelCompar
version 4.0 software (Applied Maths, Kortrijk, Belgium). After
conversion and normalization of gels, the degrees of similarity of DNA
profiles were determined by the Dice coefficient or the
maximum-correlation coefficient, and dendrograms were generated by the
unweighted pair group method using arithmetic averages. Three
computerized DNA profile libraries were generated with data obtained
from 101 strains of Salmonella serovar Enteritidis (Table 1,
collection 5), previously analyzed by PFGE with the restriction enzymes
XbaI, BlnI, and SpeI (11).
The identity of the 15 duplicated strains used previously in the
reproducibility studies of the PFGE method (Table 1, collection
4) were matched against the constructed computerized libraries to
test their ability to recognize new strains.
| |
RESULTS |
PCR fingerprinting.
In order to determine the intra- and
interassay reproducibility of PCR fingerprinting in the same
laboratory, groups of duplicate strains were analyzed. For the
intraassay study, DNA extractions and PCR procedures of the duplicates
were done at the same time, whereas the amplicons were placed in
different agarose gels. Different profiles were not obtained, although
differences in band intensity were sometimes observed. In the
interassay analysis of reproducibility, duplicates were cultivated and
their DNA was extracted and amplified at different times during the
study. In this case, some of the duplicates showed differences in band
profiles, mainly associated with faint bands with various grades of
intensity. Consequently, interassay reproducibility values were low,
ranging from 44 to 73%. To assess the intercenter reproducibility of
PCR fingerprinting, strain collection 1 (Table 1) was duplicated and
separately processed and analyzed in two laboratories (centers A and
B). The reagents and the preparation of the samples were identical in
both centers and carried out by the same person. In center A,
amplification of the DNA was carried out in a single batch with the PE
Applied Biosystems 9600 thermocycler. In center B, amplification of the DNA from the isolates was carried out in four batches, due to the
impossibility of running all the samples together in a single experiment with the Linus Autocycler 32 thermocycler, and with replicative purposes to assess interassay reproducibility. In center A, the results of the PCR fingerprints with the three-primer set showed that the most of the strains clustered in a single cluster in the dendrograms. In center B, the banding
profiles of the strains extracted and amplified in each batch clustered together. When the results obtained in both centers were
compared, a large number of discrepancies between duplicate
strains were observed, in number, intensity, and position of the bands
as is shown with the primer M13 in Fig.
1.
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FIG. 1.
Similarity dendrograms of band patterns of
Salmonella serovar Enteritidis strains produced by PCR
fingerprinting and M13 primer. (A) Amplification of DNA was performed
with a Linus Autocycler 32 thermocycler in several batches. (B)
Amplification of DNA was performed with a PE Applied Biosystems 9600 thermocycler in one batch.
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|
IRS-PCR.
The procedure of DNA extraction described by Garaizar
et al. (5) was chosen for the IRS-PCR method, because of the
poorly defined DNA bands obtained with the boiling procedure used
in PCR fingerprinting (data not shown). Optimization of the
ligation step was carried out by testing variable amounts of DNA
(range, 0.4 to 1 µg) against variable amounts of adapters (range, 15 to 30 pmol). The PCR variables were used while testing variable amounts of restricted ligated DNA (range, 0.5 to 1 µl), MgCl2
(range, 1 to 4 mM), and primers (range, 35 to 75 pmol). In the ranges studied, no significant differences in band profiles were obtained, except with MgCl2, where low concentrations inhibited
amplification. The optimal conditions are described in Materials and
Methods. Reproducibility of IRS-PCR was assessed in
duplicate strains in which their DNA were extracted and amplified
at different times. Identical band profiles were obtained, in terms of
number, intensity, and position, and therefore the reproducibility
values reached 100%.
Nine strains belonging to different serovars of
Salmonella
(Table
1 collection 2) were analyzed by IRS-PCR with a combination
of
the restriction enzymes
XbaI-
HhaI, and
XbaI-
TaqI. Amplification
of fragments was carried
out in duplicate, and identical results
were obtained. With the enzyme
combination
XbaI-
HhaI, the number
of bands
obtained ranged from 7 to 16 (Fig.
2),
and with the combination
XbaI-
TaqI, this number
ranged from 3 to 8 (data not shown). Typability
and discrimination
values between serovars of
Salmonella reached
100%. When 36 strains of
Salmonella serovar Enteritidis (Table
1,
collection 3) were processed by IRS-PCR, amplification of
the DNA from
the isolates was carried out in three batches with
the Linus Autocycler
32 thermocycler. The number of bands obtained
with the enzyme
combination
XbaI-
HhaI was 4 to 11 and divided
the
collection into four clusters as is shown in Fig.
3, obtaining
an index of discrimination
of 0.52. Interestingly, strains from
Denmark and England were
grouped together in the dendrogram at
90% similarity in
cluster A, but Spanish strains were grouped
in clusters B, C, and D. This increase in discrimination in the
group of the Spanish strains was
not corroborated by their PFGE
profile data. A subgroup of nine
strains (three for each country)
were further processed
with selective primers PX-A, PX-C, PX-G,
and PX-T. These primers
had an extension of a nucleotide at the
3' end (A, C, G, and T
respectively) of primer X (Table
2). With
selective primers, a lower
number of bands was obtained, and discrimination
between the
selected strains was not increased (data not shown).
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FIG. 2.
IRS-PCR of different serovars of Salmonella
obtained with the enzyme combination XbaI-HhaI.
(A) Lane M, molecular mass marker (pGEM). Lane 1, serovar Arizonae;
lane 2, serovar Litchfield; lane 3, serovar Virchow; lane 4, serovar
Miami; lane 5, Salmonella serogroup 11; lane 6, serovar
Abony; lane 7, serovar Virchow; lane 8, serovar Dublin; lane 9, serovar
Enteritidis; lane 10, serovar Typhimurium. Numbers on the left indicate
the size of the marker in base pairs. (B) Similarity dendrogram of the
IRS-PCR patterns.
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FIG. 3.
Similarity dendrogram of the IRS-PCR patterns of 36 epidemiologically unrelated Salmonella serovar Enteritidis
strains obtained with enzyme combination
XbaI-HhaI. En, England; Dk, Denmark; Sp, Spain.
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PFGE.
The reproducibility of the method was tested by
independently examining the same set of strains (Table 1, collection 4)
in centers B and C (Fig. 4). Table
3 shows the visual and automatic computerized analyses of PFGE profiles generated by both centers. When
visual analysis was performed, a very high agreement in intensity, number, and position of bands in most of the strains analyzed was
observed, reaching 93.33% of reproducibility value. In only one
strain, strain 15, a disagreement occurred after digestion with
XbaI and BlnI enzymes. In each case, an extra
macrorestriction band of 40 kb was found when the experiments were
carried out in center B. Therefore, two different subtype profiles were
assigned to this strain, depending on the place where the analysis was made (with the XbaI enzyme A10 or A12 and BlnI
enzyme A10 or A12 in center B or C, respectively). In contrast, only
profile A1 was found with SpeI enzyme in both laboratories
for this strain. Computerized analysis of these profiles confirmed the
high reproducibility of the PFGE method.
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FIG. 4.
Agarose gels of the PFGE profiles of
Salmonella serovar Enteritidis obtained in the intercenter
reproducibility study of PFGE. Fifteen duplicated strains were
processed in two laboratories. Pictures on the left were obtained in
center C (Denmark) and those on the right in center B (Spain). (A) DNA
digested with restriction enzyme XbaI. (B) DNA digested with
restriction enzyme SpeI. (C) DNA digested with restriction
enzyme BlnI. Lanes M, lambda ladder used as a molecular
marker. Numbers on the right indicate the size of the marker in
kilobases.
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TABLE 3.
Reproducibility of PFGE method in the analysis of 15 epidemiologically unrelated Salmonella serovar Enteritidis
strains in two laboratories; correlation between visual and
computerized analysis
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Library database systems.
On basis of the high intercenter
reproducibility values of PFGE reported here and the high
discrimination values reported previously, the PFGE method was chosen
to establish a library computerized system for Salmonella
serovar Enteritidis. For this purpose, bacterial collection 5 (Table 1)
was used. The collection was composed of 101 strains belonging to the
most prevalent phage types, isolated from human, food, and animal
sources, and were previously analyzed by PFGE (11).
Three computerized libraries of PFGE DNA profiles were constructed from
the data obtained with restriction enzymes XbaI,
BlnI, and SpeI. After library construction, the databases' abilities to recognize and analyze new profiles were
assessed when the PFGE results obtained with bacterial collection 4 (Table 1) were analyzed. The comparisons between profiles assigned automatically by the computer and visually are shown in Table 3. When
comparison with the Xba library was carried out, full agreement existed between visual and computer comparison for most of
the strains, and only two strains showed different data. Strain 15 was
automatically assigned to profile A12 as well as to profile A10 (100%
of similarity), independently of the laboratory where the
analysis was made. The profile of strain 13 was recognized as A12 with
a similarity of 92.9% by computer, whereas visual comparison assigned
the strain to profile D. Also, the results of the profile
recognition study with the Bln library showed concordant results for most strains. In this case, strain 1 was recognized as A1
as well as A8 (100% of similarity) and strain no. 15 was assigned to
profile A10 as well as to A12 (100% of similarity) by automatic
comparison, as the computer was unable to distinguish between the
profiles. When results obtained by the SpeI enzyme were
compared with to the database in the Spe library, more
difficulties were found, and the index of analysis had to be modified
from the Dice coefficient to the maximum-correlation coefficient in order to get a better concordance between visual and automatic comparisons. The low levels of similarity obtained in strain 13 revealed that these profiles were not previously recorded at the three libraries.
| |
DISCUSSION |
For the establishment of an international database of
Salmonella DNA profiles, it is necessary to build up large
databases containing patterns from a wide variety of strains,
to which unknown strains could be compared. Computerized gel
analysis allows the comparison of band profiles allocated in different
gels and the construction of databases that could be useful in
developing a library typing systems of microorganisms. These techniques
could allow communication between centers and rapid detection of the spread of pathogens and emerging infections. The objective of this
study was to explore the possibilities of establishing such a library
typing system on the basis of several molecular epidemiological techniques that allow strain characterization of Salmonella
serovar Enteritidis. As a result of the application of such
molecular techniques, a not-obvious relation between the source of the
isolates (i.e., food, human, or environmental) with their genotypes was found.
PCR fingerprinting method.
In a previous study
(13), we showed the usefulness of PCR fingerprinting
procedures in the epidemiological typing of Salmonella serovar Enteritidis. The primary objective of the present study was to
explore the possibilities of establishing a library typing system for
serovar Enteritidis on the basis of PCR fingerprinting, assessing the
intraassay, interassay, and interlaboratory reproducibility of the
method. When PCR fingerprinting was performed with the Linus Autocycler
32 thermocycler, the isolates were processed in different assays or
batches and were electrophoresed in several agarose gels. Clustering of
band profiles was clearly associated with the different assays. This
association was not detected when all the strains were processed in one
assay using the PE Applied Biosystems 9600 thermocycler. In these
interassay comparisons, important differences in intensity and band
number were observed. Following the strict criteria of Ellsworth et al.
(4), distinguishing between specific, reproducible, and
constant bands and nonreproducible bands, the reproducibility of
our method was 44%. Analyzing the profiles more permissively and not
taking into account the faint bands of some gels, reproducibility
increased to 73%. Nevertheless, when samples from which DNA was
extracted at the same time and amplified using the same reaction
mixture were compared at the intraassay studies, a reproducibility of
100% was obtained.
There are some studies designed to assess intercenter comparison of PCR
fingerprinting results. Penner et al. (
18), analyzing
the
results obtained in several laboratories, concluded that the
main
differences in banding profiles were due to the use of different
thermocyclers. van Belkum et al. (
32), analyzing 60 strains
of
Staphylococcus aureus in seven laboratories with the same
protocol,
DNA templates, and primers, obtained discordant results as
well.
Grundmann et al. (
8), analyzing PCR fingerprinting of
Acinetobacter isolates in seven laboratories, obtained
better reproducibility
using commercially available "PCR pellets"
and standardized primers.
In our study, the instability of band
profiles that we observed
with different thermocyclers could be due to
differences in the
transition between melting and annealing
temperatures. As reported
by Schweder et al. (
26), these
differences could influence the
sizes and amounts of the fragments
obtained. Nevertheless, in
our study other factors such as minor
differences in procedures
or PCR mixtures could not be discarded as
causes of variability
between band profiles. Our data have shown that
PCR fingerprinting
could be useful as an epidemiological marker when
strictly defined
conditions are maintained, isolates are processed in a
single
batch with identical reagents, and interassay or intercenter
comparisons
are not usually required. With this observation, we could
conclude
that with PCR fingerprinting no library-definitive typing
system
is feasible at
present.
IRS-PCR method.
A new typing method, such as IRS-PCR, has to
be evaluated prior to being considered valuable in epidemiological
typing. We have optimized the procedure for Salmonella
serovar Enteritidis, and the results are encouraging. Assay
reproducibility values were high, but the discrimination of the method
could not be considered elevated (D = 0.52), even
taking into consideration the high degree of clonality revealed by most
of the epidemiological typing methods of this serovar. In our search of
the literature on IRS-PCR, few studies have shown data from this
method. Mazurek et al. (15), described the method and used
the combination of XbaI-HhaI restriction enzymes
to characterize epidemiologically related and unrelated clinical
isolates of Mycobacterium avium and Mycobacterium
intracellulare, using PX-G selective primer to obtain unique
genotypes for the nonrelated strains. Riffard et al. (24)
obtained high discrimination results (D = 0.996) with
IRS-PCR, comparable to that obtained with PFGE in an epidemiological
study of Legionella pneumophila. The amplified fragment
length polymorphism (AFLP) procedure described by Vos et al.
(34) has several similar steps in common with IRS-PCR, but
the adapters used for IRS-PCR are different. In AFLP analysis, the
adapters are composed of oligonucleotides of 18 to 22 bases, while in
IRS-PCR, both adapters are composed of two chains, one 18 to 22 bases
long and the other 7 bases long. This characteristic of adapters in
IRS-PCR simplifies the method, reducing the number of bands and making
the analysis simpler. Aarts et al. (1) obtained good
discrimination with AFLP when analyzing 62 serovars of
Salmonella, although the discrimination values decreased
when analyzing strains of serovar Enteritidis. Our data demonstrated
that IRS-PCR is a rapid, simple, and reproducible method for
discriminating between Salmonella serovars, but
discriminating between strains of serovar Enteritidis with these
enzymes yielded relatively low values. The use of new combinations of
restriction enzymes to increase the strain discrimination and the
performance of interlaboratory multicenter studies should address their
possible use in definitive library typing in the near future.
PFGE.
The reproducibility of the PFGE method when analyzing
strains of serovar Enteritidis was high (93.33%). Although only some of the reagent batches, such as agarose and enzymes, and different equipment were used, nearly the same results were generated in this
interlaboratory study. The only exception was one strain that showed
different profiles after cleavage with XbaI or
BlnI. In both cases, an extra band of approximately 40 kb
was detected when the analysis was carried out in center C. This
observed difference may be due to the carriage of a plasmid, although
in previous studies, plasmid bands did not account for the restriction
fragment length polymorphism detected in PFGE experiments (12,
30). In order to corroborate the presence or absence
of plasmids in that strain, PFGE was performed without
restriction enzymes, and only the band of chromosomal DNA was observed
(data not shown). Therefore, the observed variation in PFGE profiles
could reflect a chromosomal rearrangement or a point mutation of the
chromosomal DNA during in vitro growth. We are considering the
possibility of cloning and sequencing such DNA fragments in order to
determine the molecular basis of that genotypic divergence. Careful
surveillance of changes in PFGE profiles would help in the
determination of the prevalence of such genetic events, responsible for
this biological variability of genotypes.
Similarity between DNA profiles in the bacterial duplicates was
quantified through the use of GelCompar software. Our study
highlighted
the importance of adequate normalization and selection
of the
parameters used for analysis and matching in computerized
gel analysis.
This observation correlates well with previous studies
(
7).
The software was efficient at recognizing and grouping
strains with
closely similar genotypic fingerprints, and results
were similar to
those expected from visual inspection of the
gels.
Library typing.
Once the interlaboratory study showed that the
PFGE method was reproducible, our aim was to assess the possibility of
constructing computerized libraries of DNA restriction enzyme profiles
and perform identity searches of new DNA profiles in these databases. Three libraries were constructed with data obtained from a previous analysis of 101 Salmonella serovar Enteritidis strains
isolated in three European countries (11). The PFGE profiles
of the 15 strains used for the reproducibility study were compared and
matched with these created libraries. The libraries consisted of
defined units which each represented a homogeneous electrophoretic
type, containing one or more representative profiles. For the creation of such libraries, it is important to emphasize that only gels with
identical molecular weight standards and similar image resolutions could be used. The possibility of adding a new unit to the library when
a new DNA profile appeared was open. When the PFGE profiles of these
new strains of Salmonella serovar Enteritidis were compared to the libraries, the program showed the best matching units with the
corresponding correlation in decreasing order. The program was
efficient at recognizing and comparing unknown patterns with Xba and Bln libraries with the same mathematical
coefficient but in the case of the Spe library, different
calculation methods had to be used to get better results.
The program examined in this study, GelCompar, proved to be a useful
tool for indicating the similarity between strains and
was able to
establish computerized libraries for epidemiological
comparison. As the
PFGE method has been shown to be reproducible,
discriminative, and
capable of feasible intercenter standardization,
we recommended the use
of PFGE for epidemiological comparisons,
such as constructing a typing
library, in combination with software
that allows construction of
computerized databases of DNA profiles.
The determination of the
prevalence of the genetic events that
could be responsible for
changes in PFGE profiles in this serovar
could help address the
question of the potential long-term use
of this
system.
| |
ACKNOWLEDGMENTS |
This work was supported by Research Project grant 093.123-EA
125/96 from the Basque Country University and Education, University, and Investigation Department grant PI-1998-52 from the Basque Government, Spain.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Immunology, Microbiology, and Parasitology, Basque Country
University, Apdo. 450, 01080 Vitoria-Gasteiz, Spain. Phone: 34 945 013912. Fax: 34 945 130756. E-mail:
oipgacaj{at}vc.ehu.es.
| |
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Abstract
Introduction
