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Applied and Environmental Microbiology, November 1999, p. 4830-4836, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Potential of Three-Way Randomly Amplified
Polymorphic DNA Analysis as a Typing Method for Twelve
Salmonella Serotypes
S. M.
Soto,1
B.
Guerra,1
M. A.
González-Hevia,2 and
M. C.
Mendoza1,*
Departamento de Biología Funcional,
Area Microbiología, Universidad de Oviedo, 33006 Oviedo,1 and Laboratorio de Salud
Pública, Principado de Asturias, 33011 Oviedo,2 Spain
Received 10 March 1999/Accepted 4 August 1999
 |
ABSTRACT |
The potential of a three-way randomly amplified polymorphic DNA
(RAPD) procedure (RAPD typing) for typing Salmonella
enterica strains assigned to 12 serotypes was analyzed. The
series of organisms used included 235 strains (326 isolates) collected
mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth
cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to
define RAPD types and to evaluate the procedure as a typing method at
the species and serotype levels. The typeability was 100%; the
reproducibility and in vitro stability could be considered good. The
concordance of RAPD typing methods with serotyping methods was 100%,
but some profiles obtained with two of the three primers were obtained
with strains assigned to different serotypes. The discrimination index
(DI) within the series of organisms was 0.94, and the DI within
serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and
0.66, respectively. Within these serotypes the most common RAPD types
were differentiated into phage types and vice versa; combining the
types identified by the two procedures (RAPD typing and phage typing)
resulted in further discrimination (DI, 0.96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD
typing method support the conclusion that it can be used as a tool for
identifying Salmonella organisms and as a typing method
that is complementary to serotyping and phage typing methods.
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INTRODUCTION |
Typing methods are useful tools for
performing epidemiological surveys of pathogenic bacteria. They are
used for the following two main purposes: to discriminate between
epidemiologically unrelated isolates belonging to the same microbial
species or taxon based on phenotypic or genotypic characteristics or
traits called epidemiological markers and to recognize a close
relationship among isolates derived from the same outbreak or chain of
transmission, reflecting the fact that the isolates are recent
derivatives of a simple ancestor cell (14, 17). The
usefulness of a trait for typing is related to its stability in a given
strain and its diversity in the strains forming one species. The
organisms of the genospecies Salmonella enterica
(Salmonella choleraesuis) are grouped into more than 2,300 serotypes or serovars as determined by the Kauffmann-White serological
scheme (7). However, serotyping is normally inadequate as a
single typing method for epidemiological purposes for the following two
main reasons: (i) typological cataloging of surface exposed antigens
can provide little information concerning the overall genetic
relationships of strains belonging to different serovars (9, 10,
13, 15); and (ii) most human salmonellosis episodes and
outbreaks, as well as livestock outbreaks, are caused by a few
serotypes. Over the last few years several genetic typing methods for
Salmonella spp. have been evaluated, and these methods appear to be useful tools for epidemiological and phylogenetic purposes. A rapid method that is used universally, randomly amplified polymorphic DNA (RAPD) segment analysis (21, 22) performed with different primers, has been proposed as a tool for characterizing organisms belonging to some Salmonella serovars (3, 5,
8, 10, 16).
In this paper we describe the results of a RAPD analysis performed
directly with aliquots of water-diluted overnight cultures of bacteria;
in this analysis we used three selected primers, optimized PCR
conditions, and a large series of organisms, including both
epidemiologically related and unrelated isolates assigned to 12 serotypes and collected in the Principality of Asturias (Spain).
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MATERIALS AND METHODS |
Bacterial strains.
This study was performed with 326 isolates that were grouped into 235 epidemiologically unrelated
Salmonella strains and were isolated in the Principality of
Asturias from 1984 to 1998. A total of 202 isolates (199 strains) were
associated with sporadic human salmonellosis episodes that occurred at
different times and/or in different hospitals, and another 108 isolates
(21 strains) were associated with 21 outbreaks; 4 strains were isolated
from foods, and 11 strains were isolated from water samples not
associated with outbreaks. The isolates belonged to three serogroups.
The serogroup B isolates included 73 serotype Typhimurium isolates (in
59 strains), 6 serotype Bredeney strains, 5 serotype Brandenburg strains, and 6 serotype Derby strains. The serogroup C isolates included 7 serotype Infantis strains, 18 serotype Hadar strains, 57 serotype Virchow isolates (in 20 strains), 20 serotype Ohio isolates
(in 16 strains), 4 serotype Muenchen strains, and 3 serotype Newport
strains. The serogroup D isolates included 83 serotype Enteritidis
isolates (in 54 strains) and 44 serotype Panama isolates (in 37 strains). Serotyping of organisms other than members of serotypes
Enteritidis and Typhimurium, as well as phage typing of serotype
Enteritidis, Typhimurium, and Virchow organisms (by using the methods
described by Ward et al. [20], Anderson et al.
[1], and Chambers et al. [2],
respectively) were carried out by workers at the Centro Nacional de
Microbiología, Majadahonda, Madrid, Spain. In addition, nine
strains obtained from different collections representing the three
serogroups and their most common serotypes and three outgroup strains
(Escherichia coli ATCC O111-B4, Yersinia
enterocolitica ATCC 27729, and Lactobacillus plantarum ATCC 1497) were analyzed and used as reference strains.
RAPD fingerprinting.
The assays were performed with the
following three types of DNA templates: (i) 100 ng of DNA which was
isolated with a Nucleon BACC-3 for blood and cell culture kit (Amersham
Pharmacia Biotech), (ii) 15 µl of a 10-fold distilled water dilution
of a Luria-Bertani broth overnight culture, and (iii) 15 µl from a
single colony grown on nutrient agar (the colony was picked and
resuspended in 150 µl of distilled water; the suspension was boiled
for 5 min; and the supernatant was collected after centrifugation for 2 min). RAPD reactions were carried out with the following three primers:
primer S (5'-TCACGATGCA-3'), which was described by Williams et al. (22), and primers OPB-6 (5'-TGCTCTGCCC-3')
and OPB-17 (5'-AGGGAACGAG-3'), which were described by
Lin et al. (10). In this study the latter two primers were
designated primers B and C, respectively. The conditions that were
selected as the optimal conditions for obtaining accurate amplified
band profiles with the three primers were as follows. Assays were
performed in 50-µl reaction mixtures containing an amplification
buffer (10 mM Tris-HCl [pH 8.8], 1.5 mM MgCl2, 50 mM KCl,
0.1% Triton X-100), each deoxynucleoside triphosphate (Roche
Diagnostics, Barcelona, Spain) at a concentration of 200 µM, 0.9 µM
primer (Amersham Pharmacia Biotech), 2 U of DyNAzyme II DNA polymerase (Finnzymes OY, Espoo, Finland), and 100 ng of template DNA or 15 µl
of a bacterial dilution in water. The temperature cycling program used
with a Perkin-Elmer Gene Amp PCR system (models 2400 and 9600) was as
follows: 2 initial cycles consisting of 94°C for 4 min, 35°C for 2 min, and 72°C for 2 min, followed by 35 cycles consisting of 94°C
for 30 s, 35°C for 1 min, and 72°C for 2 min and a final
extension step consisting of 72°C for 5 min. The reaction products
were analyzed by electrophoresis on 1.5% agarose gels, stained with
ethidium bromide, and photographed under UV light. Lambda DNA digested
with PstI was used as the molecular weight marker. Each
amplified band profile was defined by the presence or absence of bands
at particular positions on the gel. Profiles were considered different
when at least one polymorphic band was identified. The profiles were
labelled with the letters assigned to the primers followed by Arabic numerals.
Reproducibility was examined by comparing the band profiles obtained in
at least three RAPD analyses of representative strains that produced
each band profile. The intermethod concordance (IMC) was defined as the
maximum proportion (percentage) of strains that were grouped together
into unique types by RAPD typing and by another method (17).
The discrimination index (DI) (i.e., the probability that two unrelated
strains obtained from the population would be placed in different
typing groups) was calculated by using Simpson's index of diversity
(6).
PstI-SphI (PS) ribotyping was performed as
described by Landeras and Mendoza (8). The method used
consisted of double digestion of DNA with PstI and
SphI, hybridization with an rrn operon, and use
of a nonradioactive DNA labelling and detection kit (Roche Diagnostics).
| |
RESULTS |
Selection of primers and optimal PCR conditions.
In the first
step, primers were selected (from a set of seven aleatory
oligonucleotides) and PCR conditions were optimized in order to
maximize the discriminatory power of RAPD analysis and to test the
reproducibility, precision, and ease of interpretation of amplified
band profiles. This analysis was performed by using purified DNA and a
series of 30 Salmonella strains representing common
serotypes (serotypes Enteritidis, Typhimurium, Hadar, and Virchow) and
uncommon serotypes (serotypes Derby, Bredeney, and Muenchen), as well
as 3 outgroup strains (E. coli ATCC O111-B4, Y. enterocolitica ATCC 27729, and L. plantarum ATCC 1497).
Different concentrations of the DNA template primer and
Mg2+, different numbers of cycles (29 to 35 cycles), and
different annealing temperatures (35 to 40°C) were examined. The
primers selected were primers S, B, and C, and they were selected for three reasons: (i) under the conditions described above they yielded the most accurate amplified band profiles; (ii) the band profiles obtained with each of the primers exhibited high levels of similarity with respect to the size and number of amplified bands but differed greatly from the band profiles obtained for the three
non-Salmonella strains; and (iii) strains of different
serotypes yielded different band profiles with the three primers (data
not shown). The reproducibility of the technique when each primer was
used was initially examined by comparing the fingerprint profiles
obtained from three RAPD analyses of the 30 Salmonella
strains and 3 non-Salmonella strains.
In order to simplify the procedure, DNA templates were obtained in two
other ways: from the supernatant of a boiled colony
and from a
water-diluted overnight culture of bacteria. Under
the conditions used,
the band profiles generated by the two procedures
were the same, were
reproducible, and were the same as the results
obtained with template
DNA. Thus, for the subsequent steps, the
last option was
chosen.
RAPD analysis performed with selected primers.
The
Salmonella organisms which we studied (326 isolates and 9 reference strains) were analyzed by performing a RAPD analysis with the
primers and PCR conditions described above. With primer S, 21 amplified
DNA band profiles were differentiated; each of these profiles included
two to five fragments which were 250 to 2,000 bp long. Two fragments,
which were about 500 and 900 bp long, were found in 17 and 20 band
profiles, respectively, and in 93.5 and 99.5% of the isolates,
respectively (Fig. 1 and Table 1). Different amplified fragments
appeared to be characteristic of different serotypes (a ca. 1,800-bp
fragment for serotype Infantis, a 250-bp fragment for serotype Derby,
and a 1,900-bp fragment for serotype Virchow). Five profiles (profiles
S01, S07, S11, S12, and S15) were produced by strains belonging to
different serotypes. The strains of serotypes Enteritidis, Infantis,
and Bredeney produced single profiles (profiles S01, S11, and S04, respectively), while the serotype Typhimurium strains produced seven
profiles, the serotype Panama and Hadar strains produced three profiles
each, and the strains of serotypes Virchow, Ohio, Brandenburg, Derby,
Muenchen, and Newport produced two profiles each.
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FIG. 1.
Amplified band profiles generated with primer S for
S. enterica. Lanes A, phage  DNA digested with
PstI; lanes 01 to 21, primer S profiles. Profile S12
(representing serotype Typhimurium strain ATCC 14028) (lanes 12) was
included in both gels. The distribution of strains in serotypes and
primer S profiles is shown in Table 1.
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With primer B, 14 amplified DNA band profiles were differentiated; each
of these profiles included four to seven fragments
which were 400 to
2,500 bp long. Two fragments, which were about
400 and 2,000 bp long,
were found in 13 profiles, and a third
fragment, which was about 900 bp
long, was present in 10 profiles
and 94.72% of the isolates (Fig.
2 and Table
1). Six profiles
(profiles
B01, B02, B03, B04, B06, and B08) were produced by strains
belonging to
different serotypes. The serotype Hadar and Panama
strains produced
three profiles each; the serotype Enteritidis,
Virchow, Typhimurium,
Bredeney, Derby, and Newport strains produced
two profiles each; and
the strains of the resting serotypes each
produced a single profile.
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FIG. 2.
Amplified band profiles generated with primer B for
S. enterica. Lane A, phage DNA digested with
PstI; lanes 01 to 14, primer B profiles. The distribution of
strains in serotypes and primer B profiles is shown in Table 1.
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With primer C, 40 amplified DNA band profiles were obtained. Most of
the profiles were defined by considering only bands that
were 1,700 bp
long or smaller. The only exceptions were profiles
C27, C28, and C36,
which contained a clear band at about 3,000
bp, which was also
considered; for the rest of the profiles the
bands that were located
between 1,700 and 2,000 bp were not reproducible.
Thus, the profiles
defined included three to seven fragments which
were 200 to 3,000 bp
long. No fragment was found in all of the
profiles, but two fragments,
at about 510 and 1,700 bp, were found
in 34 and 35 profiles,
respectively, and in 90 and 96.6% of the
isolates, respectively (Fig.
3). No profile was produced by strains
belonging to different serotypes. Only serotype Infantis strains
produced a fragment (at about 3,000 bp) that was characteristic
of a
serotype. The strains belonging to 11 serotypes (all of the
serotypes
except serotype Ohio) could be differentiated with this
primer (Table
1).
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FIG. 3.
Amplified band profiles generated with primer C for
S. enterica. Lane A, phage DNA digested with
PstI; lanes 01 to 40, primer C profiles. Profile C11
(representing serotype Typhimurium strain ATCC 14028) (lanes 11) was
included in all three gels. The distribution of strains in serotypes
and primer C profiles is shown in Table 1.
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On the other hand, it is noteworthy that when a strain initially
classified as a
Salmonella strain produced a band profile
without fragments found in the other profiles, it was suspected
of not
being a
Salmonella strain and was reclassified. In all
of
these cases the strains were not
Salmonella strains.
Similarly,
when a strain initially assigned to a specific serotype
produced
(with the three primers) band profiles characteristic of
another
serotype, it was reserotyped, confirming that it had been
registered
with an erroneous
serotype.
Concordance of RAPD typing with serotyping and phage typing.
RAPD typing results were compared with serotyping results (see above)
and, for serotypes Enteritidis, Typhimurium, and Virchow, with phage
typing results. The IMC was 100% for RAPD typing and serotyping, but
the IMC could be lower with other series of organisms because strains
belonging to different serotypes sometimes produce the same profiles
(Table 1). Phage typing differentiated the serotype Enteritidis strains
(n = 42) into the following nine phage types: PT1 (6 strains); PT4 (14 strains); PT6a (10 strains); and PT5a, PT6, PT7, PT8,
PT11, and PT13a (1 strain each). Four strains were non-phage typeable
(NPT), and two other strains produced a nonrecognized lysis pattern
(NRP). Serotype Typhimurium strains (n = 59) were
differentiated into the following 15 phage types: DT104 (17 strains);
DT193 (8 strains); DT195 (2 strains); DT80 (2 strains); DT96 (2 strains); DT23 (2 strains); and DT2, DT5, DT28, DT66, DT110, DT120,
DT124, DT133, and DT204 (1 strain each). Seventeen other strains were
NPT. Serotype Virchow strains (n = 17) were
differentiated into the following eight phage types: PT4a (3 strains);
PT8 (7 strains); PT31 (2 strains); and PT16, PT17, PT19, PT33, and PT34
(1 strain each). Within the three serotypes the IMC between RAPD typing
and phage typing was low. In fact, within each serotype the most common
RAPD profiles were differentiated into several phage types, and
conversely, the most frequent phage types were differentiated into RAPD
types, with the following two relevant exceptions: the 13 strains (26 isolates) belonging to serotype Enteritidis PT4 fell into RAPD type S01
B01 C01, and seven of the eight serotype Virchow PT8 strains fell into
RAPD type S03 B04 C31 (the eighth strain differed only in the profile generated with the third primer, C34) (Table
2).
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TABLE 2.
Subdivision of serotype Enteritidis, Typhimurium, and
Virchow RAPD types into phage types and subdivision of phage types
into RAPD types
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Epidemiological concordance, in vivo stability, and correlation of
RAPD typing with PS ribotyping.
Three other criteria for RAPD
typing (epidemiological concordance, in vivo stability, and correlation
with another genetic typing system [PS ribotyping]) were examined by
comparing results obtained with seven groups of epidemiologically
related isolates. Each group included sequential isolates recovered
from a single patient or isolates collected from different patients and
foods associated with previously diagnosed outbreaks in our
laboratories. The PS ribotypes and their relationships with the other
markers are shown in Fig. 4, and the
following data are noteworthy. In group 1, three serotype Enteritidis
isolates collected from one urine sample and two feces samples from a
single female fell into RAPD profile S01 B01 C06, ribotype E-PS3, and
phage type PT6a. In group 2, four serotype Ohio isolates collected over
a 3-month period from the feces of a single child fell into RAPD
profile S05 B01 C37 and ribotype O-PS1. In group 3, three serotype
Virchow isolates collected from the feces of an infant over a 3-month period fell into RAPD profile S03 B04 C32, ribotype V-PS1, and phage
type PT4a. In group 4, serotype Enteritidis isolates collected from the
feces of nine patients and one food handler, as well as from four
contaminated foods (two cakes, soup, and lamb), associated with a
restaurant outbreak fell into RAPD profile S01 B01 C01, ribotype E-PS1,
and phage type PT4. In group 5, serotype Enteritidis isolates collected
over a 3-month period from the feces of 14 elderly people living in a
rest home fell into RAPD profile S01 B01 C01, ribotype E-PS3, and phage
type PT1. In group 6, serotype Typhimurium isolates that were
associated with a family outbreak caused by a piece of defectively
cured ham and were collected from the feces of the patients and from
the ham fell into RAPD profile S11 B03 C12 and ribotype T-PS1 and were
NPT. In group 7, serotype Typhimurium isolates that were collected from
the diarrheic feces of seven young women living in a drug
rehabilitation center and were associated with an outbreak related to
water as the infection vehicle fell into RAPD profile S12 B03 C15,
ribotype T-PS10, and phage type DT96.
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FIG. 4.
Correlation of RAPD typing with serotyping, phage
typing, and PS ribotyping for seven groups of epidemiologically related
Salmonella isolates. RAPD-S, primer S RAPD profile; RAPD-B,
primer B RAPD profile; RAPD-C, primer C RAPD profile; NP, non-phage
typeable; E, Enteritidis; O, Ohio; V, Virchow; T, Typhimurium.
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DISCUSSION |
The use of PCR-based techniques has had a revolutionary impact on
the diagnosis of infectious diseases. Because these techniques can
detect or analyze minute amounts of microbial DNA or RNA sequences, they are highly sensitive and specific methods for identifying pathogens. The RAPD fingerprinting technique, in which arbitrary oligonucleotides are used to promote DNA synthesis at low annealing temperatures in order to determine genomic diversity, is a particularly powerful typing method. Unlike traditional PCR analysis, which requires
specific knowledge of DNA sequences and the use of target-specific sequences, the RAPD technique does not require any specific knowledge of the DNA sequences of the target organisms. This makes it a flexible
tool that has great power and general applicability (3, 10, 12,
16, 17, 21, 22).
The results presented in this paper show that RAPD analysis performed
with selected primers under well-defined conditions can be used to
reproducibly amplify random fragments of DNA from Salmonella
genomes in order to differentiate between and within serotypes. The
performance of the RAPD technique was evaluated by using several
criteria, including typeability, reproducibility, typing system
concordance, epidemiological concordance, ease of interpretation of the
amplified band profiles, and discriminatory power. With the three
primers used, all of the Salmonella isolates examined could
be assigned to amplified band profile groups. With regard to
reproducibility, a weakness frequently reported for RAPD analysis
(19), three facts must be emphasized. (i) The method has
been shown to exhibit total reproducibility in distributing strains
into amplified profile groups, and it has been shown to be a very
useful tool for epidemiological purposes, which differentiates strains
assigned to different serotypes. (ii) In different experiments the
amplified band profile of a given strain could include one or more
bands that were poorly defined or were not visualized, but the profile
was still different from other profiles. The PCR conditions used in
this work minimized but did not always eliminate weak and nonconstant
amplified fragments in some of the profiles, mainly profiles obtained
with primer C. The nonconstant fragments were not used during
differentiation of amplified band profiles. (iii) There were variations
in RAPD profiles obtained in different laboratories. Moreover, each
laboratory is free to establish which amplified fragments are
considered in order to define profiles. The concordance between
serotyping and RAPD typing was 100%. However, it is important to note
that some profiles generated with primers S and B were produced by
strains belonging to different serotypes.
The discriminatory power of the method was tested in the following two
ways: (i) by considering the number of profiles generated both with
each primer separately and combining the results obtained with the
three primers (RAPD types), in both cases within the series of
organisms and within each serotype, and (ii) by calculating the DI. For
the latter analysis, epidemiologically related isolates (isolates
collected from different samples from a single patient diagnosed as
having extraintestinal or persistent-recidivant intestinal salmonellosis, as well as isolates collected from the feces and/or blood of different patients and food handlers and from foods associated with specific outbreaks) having identical traits (serotype, phage type,
PS ribotype, and RAPD type) were assigned to single strains. The 326 isolates were grouped into 235 strains (in addition, 9 reference
strains were also included). The numbers of profiles obtained with
primers S, B, and C were 21 (DI, 0.84), 14 (DI, 0.78), and 40 (DI,
0.92), respectively. By combining the profiles obtained with the three
primers, we obtained 57 RAPD types and a DI of 0.94 for the series of
organisms; it is noteworthy that the 12 serotypes were subdivided into
two or more RAPD types and that the DI for the six most common
serotypes ranged between 0.13 and 0.78. When RAPD types were combined
with phage types, further differentiation occurred, and the DI for
serotypes Enteritidis, Typhimurium, and Virchow were 0.74, 0.96, and
0.87, respectively. For this analysis the NPT and NRP strains were
considered two more different types (Table 2).
With regard to convenience, one advantage of the three-way procedure is
its rapidity; it can be performed directly with aliquots of a
water-diluted bacterial culture, and the same conditions are used with
the three primers. A second advantage is that it is easy to interpret
the resulting amplified band profiles; the profiles obtained with each
primer exhibit a certain level of similarity, and many profiles are
serotype specific, thus revealing the relatedness of
Salmonella organisms. In addition to rapidity, the procedure
is also convenient because it is accessible, flexible and easy to
perform. Other genetic procedures, including procedures based on
pulsed-field gel electrophoresis or restriction-hybridization fragment
length polymorphism, such as ribotyping and IS200 typing, have been
evaluated and proposed as typing procedures for several serotypes
(4, 8, 11-13, 18), but they are more complex, costly, and
technically demanding than RAPD typing.
| |
ACKNOWLEDGMENTS |
We thank M. A. Usera and the Centro Nacional de
Microbiología for providing the serotyped and phage-typed
Salmonella strains and the personnel of the microbiology
laboratories of the Hospital Central de Asturias (Oviedo, Spain), the
Hospital San Agustin (Avilés, Spain), the Hospital de Jarrio, the
Hospital Cabueñes (Gijón, Spain), and the Hospital Carmen y
Severo Ochoa (Cangas del Narcea, Spain) for providing clinical isolates.
This work was supported by a grant from the Fondo de
Investigación Sanitaria (Ref. 98/0296). S. M. Soto was the
recipient of a grant from the Ministry of Culture and Education of
Spain (F.P.I./AP98/09429078).
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FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Biología Funcional, Area Microbiología, Facultad de
Medicina, C/ Julián Clavería s/n, 33006 Oviedo, Spain.
Phone: 34-985103560. Fax: 34-985103148. E-mail:
camf{at}sauron.quimica.uniovi.es.
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Applied and Environmental Microbiology, November 1999, p. 4830-4836, Vol. 65, No. 11
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Abstract
Introduction
