J Med Microbiol 55 (2006), 365-373; DOI: 10.1099/jmm.0.46257-0
© 2006 Society for General Microbiology
ISSN 0022-2615
Detection of virulence determinants in clinical strains of Salmonella enterica serovar Enteritidis and mapping on macrorestriction profiles
Sara M. Soto1,2,
Irene Rodríguez1,
M. Rosario Rodicio1,
Jordi Vila2 and
M. Carmen Mendoza1
1 Departamento de Biología Funcional, Área de Microbiología, Universidad de Oviedo, C/Julián Clavería 6, 33006 Oviedo, Spain
2 Servei de Microbiología, Centre de Diagnòstic Biologic, Hospital Clinic-IDIBAPS, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain
Correspondence
M. Carmen Mendoza
cmendoza{at}uniovi.es
Received 22 July 2005
Accepted 21 December 2005
A total of 80 strains of Salmonella enterica serovar Enteritidis, causing gastroenteritis (G) or bacteraemia (B), and three control strains (C), were subjected to: (i) detection of 14 chromosomally and 1 plasmid-located virulence genes by PCR, (ii) detection of DNA polymorphisms by XbaI and BlnI PFGE, and cluster analysis, (iii) mapping of the 15 screened sequences on macrorestriction profiles and (iv) comparison of the screening and mapping results with data available for other Salmonella strains. Identical virulence genotypes and very similar macrorestriction profiles were shown by most S. Enteritidis strains. However, a number of B strains belonged to genomic types with polymorphisms affecting fragments carrying (SPI2-slyA), (SPI2-slyA-phoP/Q-agfA), (SPI4 and/or stn) and spvC. The information obtained provides the basis for further studies on the genetic background of virulence and the molecular epidemiology of S. Enteritidis.
Abbreviations: SPI, Salmonella pathogenicity island.
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INTRODUCTION
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Nontyphoidal Salmonella serovars are one of the leading causes of food-transmitted infections in developed countries. In most cases, infection results in gastroenteritis, usually a self-limiting illness. However, bacteria can also spread beyond the intestine, and cause systemic infections involving the bloodstream and/or various body organs. To cause disease, Salmonella relies on multiple virulence factors, many of which are clustered within Salmonella pathogenicity islands (SPIs) (Marcus et al., 2000; Fierer & Guiney, 2001; Hensel, 2004). SPI1 and SPI2 each encode a structurally and functionally different type III secretion system, which is responsible for the delivery of effector proteins into the host cell cytosol. SPI3 is necessary for survival within macrophages and growth in low Mg2+ environments during the systemic phase of the disease. SPI4 is suspected to be required for intramacrophage survival and may also contribute to toxin secretion. Like SPI1, SPI5 appears mainly associated with enteropathogenesis, being involved in inflammation and chloride secretion. Some of the genes not residing in SPIs that also play a role in disease are slyA, phoP/Q, agfA and stn, encoding a transcriptional regulator of virulence genes required for survival within macrophages (initially identified as salmolysin) (Watson et al., 1999), a two-component global transcriptional regulator of Salmonella virulence (Miller, 1991), the thin aggregative fimbriae (Collinson et al., 1996a, b) and an enterotoxin (Prager et al., 1995), respectively. Furthermore, many Salmonella serovars harbour virulence (V) plasmids with variable size, depending on the serovar (Rotger & Casadesús, 1999; Marcus et al., 2000; Fierer & Guiney, 2001). All V plasmids share a highly conserved 8 kb region with five genes designated spvRABCD (Salmonella plasmid virulence). The spv region appears to promote rapid growth and survival of Salmonella within the host cells, being important for systemic infection in experimental animals.
During the last two decades, Salmonella enterica serovar Enteritidis has surpassed Salmonella enterica serovar Typhimurium as the most common serovar reported in European countries, including Spain (http//www.eurosurveillance.org). In addition to gastroenteritis, the incidence of bacteraemia caused by this serovar has also increased and, nowadays, it is far more often associated with invasive infections than serovar Typhimurium (Threlfall et al., 1992; Le Bacq et al., 1994; Rodríguez et al., 1998; Ispahani & Slack, 2000). In the present work, S. Enteritidis strains representing the most frequent or most distinct types (based on random amplified polymorphic DNA typing, antimicrobial resistance, and plasmid and integron profiles), which have been causing gastroenteritis or bacteraemia in Asturias (Spain), were further characterized by virulence-gene profiling, focusing on virulence determinants that have been shown to be relevant for S. Typhimurium pathogenesis, and by macrorestriction-PFGE analysis, using two different endonucleases (XbaI and BlnI). The detected V genes were then mapped on the macrorestiction profiles, and their location compared with data available for other Salmonella strains (Collinson et al., 1996b; McClelland et al., 2001).
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METHODS
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Bacterial strains.
A total of 3 control strains and 80 clinical isolates of S. Enteritidis were used in this study (Table 1
). Each clinical isolate could be considered as a strain since they had been recovered from different patients, at different times, and no epidemiological relationship had been recorded among patients. A total of 40 strains were recovered from faeces of patients with gastroenteric episodes (G group), and another 40 from the blood of bacteraemia patients (B group). They represented the most frequent or distinct types involved in human salmonellosis, recorded in Asturias, Spain, over the last decade (Landeras & Mendoza, 1998; Rodríguez et al., 1998; Soto et al., 2003; unpublished data).
PCR-based procedures.
Aliquots of Luria–Bertani (LB) broth overnight cultures of the strains were the source of template DNA. Primers for SPIs were designed by computerized analysis of the sequences recorded under the accession numbers or references listed in Table 2, following criteria such as gene specificity, number of base pairs, annealing temperature and size of the amplification product. Primers for other screened genes have been reported previously (Doran et al., 1993; Guerra et al., 2000). In total, 15 primer pairs (Table 2) were used either alone or in the following combinations (for multiplex PCR): set I (orgA, ssaQ, misL, spi4D and pipA), set II (invE/A, ttrC, mgtC, spi4R and agfA) and set III (sopB, slyA, phoP/Q, stn and spvC). Assays were performed in 50 µl reaction mixtures containing 2 µl template DNA, 200 µM each dNTP (Roche Diagnostics), 1 µM each primer (Sigma-Genosys), 2 U DyNAzyme II DNA polymerase and amplification buffer (Finnzymes). Amplifications were carried out in a Perkin-Elmer GeneAmp System (Model 9700) using the following program: a hot start cycle of 94 °C for 5 min, then 30 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 2 min, ending with a final extension step of 72 °C for 5 min. Aliquots (15 µl) of the amplification products were analysed by electrophoresis on 2 % agarose gels using TAE (Tris-acetate/EDTA) as running buffer (Sambrook & Russel, 2001). Gels were stained with ethidium bromide and visualized with UV light.
Macrorestriction-PFGE and hybridization.
Total DNA from S. Enteritidis strains was analysed by macrorestriction-PFGE using the CHEF-DRIII SYS220/240 (Bio-Rad) with a standard protocol (Peters et al., 2003). Briefly, Salmonella was cultured in LB broth and agarose plugs were prepared with the cells. DNA was digested with XbaI (10 U, 4 h, 37 °C; Takara Biomedicals) and the fragments generated were resolved by electrophoresis in 1·2 % agarose gels run in 0·5xTBE (Tris/borate/EDTA) buffer (Sambrook & Russel, 2001). DNA from strains displaying different XbaI profiles was also digested with BlnI (20 U, overnight, 37 °C; Takara Biomedicals). The running conditions were 200 V at 14 °C for 20 h. The included angle was 120°, and initial and final switch times were 2 s and 64 s, respectively. After electrophoresis the gels were stained with ethidium bromide and visualized with UV light. Relationships among XbaI profiles were established on the basis of the number of non-matching bands and the genetic similarity determined by the unweighted pair group method with arithmetic means (UPGMA) and the Jaccard's similarity coefficient (S) using the software program MVSP 3.1 (multivariate statistics package for PCs; RockWare).
XbaI profiles differing in one or more fragments, and BlnI profiles from all strains analysed, were transferred onto membranes and hybridized with selected probes for the genes indicated, as described by Sambrook & Russel (2001). The DNA probes were obtained by PCR from the type strain S. Typhimurium LT2 using the primers displayed in Table 2
and the commercial PCR DIG labelling mix (Roche Diagnostics). Firstly, hybridizations were performed with individual probes, in order to localize the 15 selected genes on specific fragments of the different profiles. Secondly, two combinations of selected probes, mapping on clearly distinguishable fragments, were used to hybridize single membranes. In this way, all hybridization results could be compiled into two panels (see below).
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RESULTS
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PCR-detection of virulence sequences in clinical strains of S. Enteritidis
The 80 clinical and 3 control strains of S. Enteritidis (Table 1) were tested for the presence of 15 V genes (10 of them representative of the 5 SPIs, 4 unclustered genes and 1 belonging to pENT, the V plasmid of S. Enteritidis) by conventional PCR using the single primer pairs shown in Table 2. Note that for each SPI two primer pairs were assayed, each targeting a specific internal sequence located close to one end of the SPI (Marcus et al., 2000). All strains tested generated the expected amplicons for the 14 chromosomally located V sequences (not shown), showing the presence of the 5 SPIs and the 4 unclustered V genes under study. However, two G strains and two B strains (Table 1) were negative for the plasmid gene spvC. In order to simplify the detection method, several multiplex PCRs, with different combinations of primers, were performed. Among those tested, three combinations (sets I to III, see Methods) were selected, because they generated well-defined profiles and faithfully reproduced the results obtained with individual primers (Fig. 1).
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Fig. 1. PCR detection of Salmonella virulence genes. Gel lane L, DNA ladder (100 bp; Gibco BRL); gel lanes I–III, multiplex PCR using the primer sets I (spi4D-1231 bp, misL-986 bp, ssaQ-677 bp, orgA-540 bp, pipA-406 bp), II (spi4R-1269 bp, ttrC-920 bp, mgtC-655 bp, invE/A-500 bp, agfA-261 bp) and III (sopB-1170 bp, slyA-700 bp, stn-617 bp, spvC-424 bp, phoP/Q-299 bp).
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Macrorestriction analysis of S. Enteritidis strains
The 83 S. Enteritidis strains were first analysed by XbaI restriction of the genome, and fragment separation by PFGE. This procedure reveals polymorphisms in the total bacterial DNA, including the chromosome and plasmids larger than 15 kb. A total of 10 different XbaI profiles (X1–X10), with fragments ranging from 19 to 900 kb in size (Fig. 2a, b) were found. Each of the control strains generated a distinct profile: X1, X2 and X3 for ATCC 13076, CNM PT4 and CNM PT6a, respectively, while the clinical strains could be distributed into nine profiles (X2–X10), although only X2 and X3, the two prevalent profiles, included both G and B strains (Table 1, Fig. 2a). The 10 profiles shared a total of 9 matching and 16 non-matching bands (some formed by 2 or 3 unresolved fragments). The similarity between profiles was higher for G strains (
85 %) than for B strains (66 %).
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Fig. 2. XbaI macrorestriction analysis of S. Enteritidis and mapping of V genes. (a) PFGE profiles generated from representative strains by XbaI digestion: (from left to right) X1, ATCC 13076; X2, CNM PT4; X3, CNM PT6a; X2, LSP 34/00; X3, 49/00; X4, LSP 98/00; X5, LSP 230/00; X6, LSP 524/00; X7, LSP 108/01; X8, LSP 376/95; X9, LSP 421/95; X10, LSP 141/97. A to P (on the left) indicate the fragments identified in the XbaI restriction map of SSU7998 (Liu et al., 1993), which coincide with those in ATCC 13076 (X1 profile). (b) Dendrogram of similarity illustrating the genetic relationships between the XbaI profiles shown in (a). (c, d) Hybridization of the DNA from (a) with the probes shown on the left. Bold numbers indicate the fragments absent in SSU7998 (Liu et al., 1993). a–f, fragments in which other probes also mapped: a, pipA; b, orgA; c, spi4D; d, ttrC, slyA; e, phoP/Q; f, mgtC. L, lambda ladder PFGE Marker (New England BioLabs).
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Interestingly, the X1 profile, only generated by the control ATCC 13076, apparently coincided with the XbaI PFGE profile of S. Enteritidis SSU7998, for which the XbaI genomic cleavage map has been constructed (Liu et al., 1993). Two differences between X1 and the remaining XbaI profiles are remarkable: (i) the presence in X1 of 250 and 76 kb fragments (labelled C and G in the SSU7998 XbaI restriction map), which were absent from the other nine profiles, and (ii) the presence in the latter of an approximately 265 kb fragment, absent from X1.
For an accurate mapping of V genes on the S. Enteritidis genome (see below), one strain representative of each of the XbaI profiles was also subjected to macrorestriction analysis with BlnI, a second endonuclease for which the physical map of S. Enteritidis SSU7998 is available (Liu et al., 1993). In this case, six different BlnI profiles (B1–B6) were detected (Fig. 3a). It should be noted that, although the X1 profile apparently coincided with that of SSU7998, none of the BlnI profiles matched exactly the one reported for this strain. In fact, the B1 profile generated by the only X1 strain (ATCC 13076) differed from the BlnI profile of SSU7998 (Liu et al., 1993) in that it lacked the F662 fragment of the latter strain, showing instead two fragments of 360 and 300 kb. In addition, it also lacked an approximately 305 kb fragment present in all of the remaining profiles.
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Fig. 3. BlnI macrorestriction analysis of S. Enteritidis and mapping of V genes. (a) PFGE profiles generated from representative strains by BlnI digestion: (from left to right) B1-X1, ATCC 13076; B2-X2, CNM PT4; B2-X3, CNM PT6a; B2-X2, LSP 34/00; B2-X3, 49/00; B2-X4, LSP 98/00; B2-X5, LSP 230/00; B3-X6, LSP 524/00; B4-X7, LSP 108/01; B5-X8, LSP 376/95; B3-X9, LSP 421/95; B6-X10, LSP 141/97. A to L (on the left) indicate the fragments of the BlnI restriction map of SSU7998 (Liu et al., 1993). Fragments labelled ‘a’ are those not generated by SSU7998, which instead contains a F662 (662 kb) fragment. (b) Hybridization of the DNA from (a) with the probes shown on the left. Bold numbers indicate the fragments absent from SSU7998 (Liu et al., 1993). Fragments labelled ‘b’ are those in which the slyA, phoP/Q and agfA probes also mapped. L, lambda ladder PFGE Marker (New England Biolabs).
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Mapping virulence sequences on macrorestriction genomic profiles of S. Enteritidis
The mapping of virulence determinants was achieved by Southern blot hybridization of membranes containing the different XbaI DNA profiles with probes for the 15 V genes (Fig. 2c, d). Concerning the SPIs, all profiles generated from C and G strains, and all except two profiles (X8 and X10) generated from B strains, carried the (invE/A-orgA), (ttrC-ssaQ), (mgtC-misL), (spi4R-spi4D) and (sopB-pipA) sequences on matching fragments P670/H656, C250/B245, K170, M300/N286 and A900 (numbers correspond to sizes in kb), respectively. In addition, the probes for SPI2 mapped on a non-matching fragment of 186·5 kb in the X8 profile, and the probes for SPI4 on a non-matching fragment of 485 kb belonging to X10. In both profiles, probes for the remaining islands mapped on the matching fragments indicated above. With regard to other chromosomal V genes: (i) the slyA probe mapped apparently on the same fragments as SPI2, (ii) the probes for phoP/Q and agfA mapped on the approximately 265 kb fragment common to X2–X10, and on one of two alternative fragments [C250 (250 kb) or B245 (245 kb)] in X1, and (iii) the stn probe mapped on the same fragments as SPI4 in all profiles except X8, where it was located on a 356 kb fragment. The spvC probe mapped on a 60 kb fragment, the size expected for pENT, in seven of the eight XbaI profiles generated by strains that proved to be spvC positive by PCR amplification. In the remaining profile (X7, only generated by one B strain), spvC mapped on a non-matching fragment of approximately 67 kb.
With the data presented above, only SPI3 and SPI5 could be precisely located on defined fragments, labelled K170 and A900 in the SSU7998 XbaI map (Liu et al., 1993). Nevertheless, the accurate position of the remaining genes could be established by hybridization of selected probes (invE/A, ssaQ, spi4R, phoP/Q, stn, slyA, agfA and spvC) with the different BlnI profiles (Fig. 3b). In these experiments, (i) the invE/A probe (indicative of SPI1) mapped on fragment H684, common to all profiles, (ii) the ssaQ (SPI2), slyA, phoP/Q and agfA probes mapped on E595 (which overlaps XbaI fragments C250 and B245) in all profiles except B5 (X8), where they mapped on a non-matching fragment of 562 kb, (iii) the spi4R probe (SPI4) mapped on L268 in all except the B6 (X10) profile, where it mapped together with stn on a 476 kb fragment, (iv) the stn probe mapped on K286 in all of the remaining profiles, except B5 (X8), where it mapped on a 380 kb fragment, and (v) the spvC probe gave an identical hybridization pattern with BlnI as with XbaI. Results of these and the former hybridizations are compiled in Table 3.
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Table 3. Mapping of probes for virulence determinants on XbaI and BlnI macrorestriction profiles of Salmonella serovar Enteritidis
The hybridizations are shown in Figs 2 and 3. The distribution of strains in XbaI macrorestriction profiles is indicated in Table 1. ND, Not determined.
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DISCUSSION
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In this investigation, S. Enteritidis strains involved in either gastroenteritis or bacteraemia were screened for virulence factors known to be relevant for pathogenesis in the better characterized S. Typhimurium LT2. By conventional and multiplex PCR, it was demonstrated that the two genes used as indicators for each of five SPIs, as well as four chromosomally located V genes (slyA, phoP/Q, agfA and stn), were present in all strains, independently of the outcome of infection. However, the spvC gene of pENT was absent from four strains, two causing gastroenteritis (identified as phage type PT6a, carrying a 40 kb tem1 plasmid and generating X6/B3 macrorestriction profiles) and two involved in bacteraemia (identified as PT4, plasmid free and assigned to X9/B3). It was also shown that a B strain carried an approximately 67 kb spvC plasmid. In our laboratory, this gene has been previously detected in the majority of S. Enteritidis strains tested, but always as part of the typical 60 kb V plasmid (Rodríguez et al., 1998; Soto et al., 2003).
With regard to the applied methodology, the advantage of multiplex PCR versus conventional PCR, for the detection of specific DNA sequences, was clearly shown. Fifteen different V genes could be simultaneously tested by only three PCR reactions. Accordingly, multiplex PCR can be efficiently used to explore the pathogenic potential of a certain bacterium. In further studies, additional multiplex PCR reactions could be designed to screen not only for additional V genes, but also for other genes of interest, such as those involved in antimicrobial drug resistance.
As in other S. Enteritidis studies, macrorestriction analysis with XbaI and BlnI showed a limited genomic heterogeneity in the analysed strains, and identical or similar XbaI profiles to those described here have been previously reported in clinical isolates collected in other European (Ridley et al., 1998; Lukinmaa et al., 1999; Garaizar et al., 2000; Peters et al., 2003) and non-European countries (Ahmed et al., 2000; Su et al., 2002; Bakeri et al., 2003). According to similarity coefficients and the number of non-matching fragments, all profiles generated from G strains were closely related, and could then be considered as members of a single XbaI restriction cluster (S84 % and 
4 non-matching fragments). Moreover, all differences observed between profiles of the G group were due to fragments smaller than 100 kb, which could be identified as extrachromosomal DNA. One of them was assigned to pENT, and the other two to R plasmids (as confirmed by hybridization of a tem1 probe with an approximately 40 kb fragment, common to the X3, X5 and X6 profiles, and with an approximately 90 kb fragment in the X4 profile; unpublished results). In contrast, the B strains showed a higher DNA polymorphism (S66 % and 11 non-matching fragments), and could, therefore, belong to more than one genomic cluster. Additionally, XbaI profiles generated by B strains included six non-matching fragments larger than 170 kb, which are probably of chromosomal origin.
It is also noticeable that using the PFGE standard conditions proposed for XbaI macrorestriction analysis of Salmonella (Sambrook & Russel, 2001), three band groupings present in S. Enteritidis profiles (with sizes of 670–656 kb, 330–286 kb and 250–240 kb), in which several virulence probes mapped, cannot be clearly separated. However, well-defined fragments were observed after BlnI macrorestriction analysis of strains representative of the different XbaI profiles. Taking the physical map of SSU7998 for the XbaI and BlnI endonucleases as a guide (Liu et al., 1993), hybridization experiments performed here mapped SPI1 to SPI5 on defined fragments of the restriction map of SSU7998 (XbaI-H656/BlnI-H684, XbaI-C255-B245/BlnI-E595, XbaI-K170, XbaI-N300/BlnI-L268 and XbaI-A900), respectively. They were categorized as matching fragments in most XbaI and BlnI profiles, including all those generated from G strains. In contrast, probes for SPI2 and SPI4 mapped on non-matching XbaI fragments of approximately 186·5 and 485 kb only present in B strains. It is also noticeable that probes for individual V genes mapped together with SPI genes on specific fragments. This was for instance the case for slyA, phoP/Q, agfA and SPI2, which were located on the same BlnI fragment in all profiles, whereas stn and SPI4 mapped on the same or adjacent fragments. In a published work (Collinson et al., 1996b), four fimbrin-encoding genes were mapped on the XbaI-BlnI genomic restriction maps of two S. Enteritidis strains (SSU7998 and 27655-3b) and S. Typhimurium LT2. As in the present work, agfA was placed on XbaI fragments B or C, and on BlnI fragment E of SSU7998. More precisely, agfA was located between centisomes 40 and 43·3, close to the break point of the 815 kb DNA inversion that distinguishes S. Enteritidis from S. Typhimurium (Collinson et al., 1996b). According to the results presented here, the phoP/Q, slyA and SPI2 genes could also be located within the inverted DNA. Within the limits of our mapping experiments, the location of other V determinants roughly coincided in the genome of S. Enteritidis and S. Typhimurium LT2 (McClelland et al., 2001), thus supporting an identical order in both serovars, as was previously reported for many other genes (Liu et al., 1993).
Taken together, results derived from the present investigation provide the basis for further studies on the genetic background of virulence and the molecular epidemiology of S. Enteritidis.
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ACKNOWLEDGEMENTS
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We thank the personnel of the Microbiology Laboratories of the Hospital Clinic (Barcelona) for their hospitality towards S. M. Soto; the Centro Nacional de Microbiología for the S. enterica serovar Enteritidis control strains; the Laboratorio de Salud Pública (LSP) (Oviedo) for the clinical strains; and the Hospital Central de Asturias (Oviedo), Hospital San Agustín (Avilés), Hospital de Jarrio and Hospital de Cabueñes (Gijón), and Hospital Carmen and Severo Ochoa (Cangas de Narcea) for their invaluable collaboration with the LSP in registering clinical isolates of Salmonella. This work has been supported by a grant from the Fondo de Investigación Sanitaria (02/0172). S. M. Soto has been the recipient of a grant from the Formación de Profesorado Universitario (AP98) of the Spanish Ministry of Culture and Education. I. Rodríguez was the recipient of a grant from the Fundación para el Fomento en Asturias de la Investigación Científica Aplicada y la Tecnología (FICYT; BP04-086).
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INTRODUCTION
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