Journal of Cystic Fibrosis 3 (2004) 165 – 172 www.elsevier.com/locate/jcf
Exopolysaccharides produced by Burkholderia cenocepacia recA lineages IIIA and IIIB Luigi Chiarini a, Paola Cescutti b,*, Laura Drigo b, Giuseppe Impallomeni c, Yury Herasimenka b, Annamaria Bevivino a, Claudia Dalmastri a, Silvia Tabacchioni a, Graziana Manno d, Flavio Zanetti e, Roberto Rizzo b a Unita` Biotecnologie, C.R. Casaccia, ENEA, 00060 Rome, Italy Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universita` di Trieste, via L. Giorgieri 1, 34127 Trieste, Italy c Istituto di Chimica e Tecnologia dei Polimeri, CNR, Viale A. Doria 6, I-95125, Catania, Italy d Laboratorio di Ricerca e Diagnostica Infettivologica, Dipartimento di Pediatria e Laboratorio Patologia Clinica, Istituto G. Gaslini, Genova, Italy e Eurand, Area Science Park, Trieste, Italy b
Received 29 December 2003; accepted 21 April 2004 Available online
Abstract Clinical and environmental strains of Burkholderia cenocepacia belonging to the recA lineages IIIA and IIIB were examined for exopolysaccharide (EPS) production. The exopolysaccharides structure was determined using mainly gas chromatography coupled to mass spectrometry and NMR spectroscopy. All the strains produced Cepacian, a highly branched polysaccharide constituted of a heptasaccharide repeating unit, composed of one rhamnose, one glucose, one glucuronic acid, one mannose and three galactose residues. This polymer is the most common exopolysaccharide produced by strains of the Burkholderia cepacia (Bcc) complex. One clinical strain produced also another polysaccharide constituted of three galactose units and one 3-deoxy-D-manno-2-octulosonic acid residues, a polymer that was previously isolated from two strains of B. cepacia genomovar I and B. cenocepacia IIIA. D 2004 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Burkholderia cenocepacia; recA lineages; Exopolysaccharide; Structure
1. Introduction Burkholderia cepacia complex (Bcc) is an important group of opportunistic human pathogens in patients affected by cystic fibrosis (CF) or chronic granoulomatous disease. Among the nine species or genomovars included in the Bcc, Burkholderia cenocepacia (previously known as B. cepacia genomovar III) [1] accounts for the majority of isolates from CF patients, comprising the most virulent and transmissible strains, often associated with a poor clinical course and high mortality of CF patients. Based on the polymorphism of the recA gene, recent papers showed that B. cenocepacia is genetically highly heterogeneous being composed of at least four phylogenetic
* Corresponding author. Tel.: +39-040-5583685; fax: +39-040-5583691. E-mail address:
[email protected] (P. Cescutti).
lineages (IIIA, IIIB, IIIC, and IIID) [2,3]. However, a worldwide distribution among CF patients has been established so far only for the lineages IIIA and IIIB. They are responsible for the majority of infection cases [3], with strains belonging to the lineage IIIA characterised seemingly by a higher transmissibility and mortality rate than the IIIB strains [4]. Little is known about the pathogenic determinants of this bacterium but some putative virulence factors have been listed. These include iron-chelating siderophores, pili, outer-membrane proteins, extracellular enzymes, intracellular survival, lipopolysaccharides, and exopolysaccharides (EPS). Bacterial EPSs are established virulence factors that act favouring evasion of host defence mechanisms, bacterial adhesion, and resistance to antibacterial agents. Recently, Chung et al. [5] observed that a virulent variant of a strain of B. cenocepacia showed an increase in EPS biosynthesis, suggesting an association between abundant EPS production
1569-1993/$ - see front matter D 2004 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jcf.2004.04.004
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and persistence of the microbe in the murine host. However, at the moment, it is still not clear whether EPS produced by Bcc has a functional role similar to that of the alginate produced by mucoid isolates of Pseudomonas aeruginosa [6]. It was demonstrated by different laboratories that B. cepacia mostly synthesises one type of EPS constituted of a highly branched heptasaccharide repeating unit (Fig. 1, Formula 1) [5,7 –10]; due to its high recurrence, it was proposed to name it Cepacian [11]. It was also observed that Cepacian is often co-produced with other polysaccharidic species [9]. On the other hand, two clinical strains isolated from CF patients attending the Cystic Fibrosis Center in Trieste (Italy) produced a polysaccharide totally different from Cepacian (Fig. 1, Formula 2) [9,12]. The correlation between Bcc species and type of EPS produced is not feasible at the moment, since generally the studies of the chemical structure of EPSs were not supported by the determination of the microbial species. Only recently, EPSs produced by a number of clinical strains with known species status were more systematically investigated [9]. The identification of as much as nine species within the Bcc raised the question whether all the species produce the same type of EPS. Furthermore, a comparison between strains of environmental and clinical origin has never been comprised in such studies, although it is generally accepted that
infections of CF patients by Bcc may occur by direct acquisition of Bcc strains from the natural environment. In an effort to gain a more complete and reliable picture of the type of EPS synthesised by the two most diffused recA lineages (IIIA and IIIB) of B. cenocepacia, EPSs produced by strains isolated from CF patients (IIIA and IIIB strains) and from the rhizosphere of maize (IIIB strains) were studied. Their structure was determined using mainly gas chromatography coupled to mass spectrometry and NMR spectroscopy.
2. Materials and methods 2.1. Bacterial strains, identification, and RAPD fingerprinting Six clinical isolates were obtained from the sputum of CF patients [13] attending the Gaslini Hospital (Genoa, Italy) and three environmental isolates were recovered from the rhizosphere of maize [14] in three different sites located in Northern, Central and Southern Italy (Table 1). Identification of isolates was accomplished according to the procedure previously described [2]. Each isolate was genetically typed by random amplified polymorphic DNA (RAPD) analysis as described by
Fig. 1. Formulas of the four exopolysaccharides produced by B. cepacia complex.
L. Chiarini et al. / Journal of Cystic Fibrosis 3 (2004) 165–172 Table 1 Bacterial strains Strain
Origin
recA lineage
RAPD type
FC30 FC42 FC52 FC24 FC75 FC87 MDIIP144 MDIIR129 MVPC12
sputum of CF patient sputum of CF patient sputum of CF patient sputum of CF patient sputum of CF patient sputum of CF patient rhizosphere of maize rhizosphere of maize rhizosphere of maize
IIIA IIIA IIIA IIIB IIIB IIIB IIIB IIIB IIIB
I I II III IV V VI VII VIII
Mahenthiralingam et al. [15]. RAPD fingerprints were compared by eye and computer software (Kodak 1D Image Analysis Software, NY). Reproducibility was verified by RAPD fingerprinting each isolate at least four times in independent experiments. Different isolates exhibiting genetic fingerprints differing by no more than two bands were considered genetically related and, thus, belonging to the same RAPD type. Isolates producing genetic fingerprints that did not match others within the bacterial population were designated as unique. 2.2. Isolation and purification of the exopolysaccharides Bacterial cells were grown for 4 or 5 days at 30 jC on a solid medium containing 2 g of yeast extract, 20 g of mannitol and 15 g of bacto agar per litre (medium MM). The bacteria were harvested by scraping the agar plates using a 0.9% NaCl solution (about 5 ml for each plate). To the obtained suspension, phenol was added at a final concentration of 5%. After stirring at 4 jC for 5 h, the cells were removed by centrifugation. The EPS was precipitated from the supernatant by addition of 4 volumes of isopropanol and then the precipitated material was dissolved in water. The procedure was repeated three times. The EPS was dialysed first against 0.1 M NaCl and then against water, the pH adjusted to neutrality and the polymer was recovered by freeze-drying. When nucleic acids and proteins were present, they were removed by treatment with DNAse, RNAse and proteinase. 2.3. General analytical methods The colorimetric phenol –sulphuric acid assay [16] was used to determine the neutral sugars present in the samples, while the test developed by Blumenkrantz and AsboeHansen [17] was applied to the colorimetric determination of uronic acids. Analytical GLC was performed with an AutoSystem XL (Perkin Elmer) gas chromatograph equipped with a flame ionisation detector, using He as the carrier gas. GLC-MS analyses were carried out on a Hewlett-Packard 5890 gas chromatograph coupled to a Hewlett Packard 5971 mass selective detector.
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Hydrolysis of the EPSs was carried out in 2 M CF3CO2H at 125 jC for 1 h. The O-acetyl groups were removed by treatment with 0.01 M NaOH at room temperature for 5 h [18]. 2.4. Composition analysis of exopolysaccharides Alditol acetates were prepared as described previously [19] using inositol as internal standard. The derivatives were separated by GLC using a SP2330 capillary column (Supelco 30 m) and the temperature program: 1 min at 200 jC, 200 –245 jC at 4 jC/min, 20 min at 245 jC. Methanolysis was performed with 1 M HCl in methanol (Supelco) at 85 jC for 18 h according to Dudman et al. [20]. Trimethylsilyl derivatives were obtained incubating the mixture of methyl glycosides with Sylon HTP kit (HMDS + TMCS + Pyridine, 3:1:9, Supelco) at room temperature for 1 h. The products were dried under a stream of N2, dissolved in n-hexane, centrifuged to remove insoluble materials and the supernatant was dried under a stream of N2. The mixtures of derivatives were separated on a HP1 column (Hewlett-Packard, 50 m) using the following temperature program: 1 min at 150 jC, 150 –280 jC at 3 jC/ min, 20 min at 280 jC. The absolute configuration of the sugar residues was established via GLC analysis of the trimethylsilylated (+)-2butyl glycosides [21,22], which were separated on a HP1 column (Hewlett-Packard, 50 m) using the temperature program: 1 min at 135 jC, 135– 240 jC at 1 jC/min, 20 min at 240 jC. 2.5. Linkage analysis The samples were sonicated to reduce the molecular weight and exchanged into the protonated form prior to chemical reactions. Methylations were performed according to Dell [23] but using potassium methylsulfinylmethanide [24]. After methylation, the permethylated samples were purified on Sep-Pak C18 cartridge, [25] prior to hydrolysis, and derivatisation into alditol acetates. The products were analysed by GLC and GLC-MS using an SP2330 capillary column (Supelco, 30 m), and the temperature program: 1 min at 150 jC, 150 –250 jC at 4 jC/min, 20 min at 250 jC. Molar ratio values were corrected by use of effective carbon-response factors [26]. 2.6. 1H-NMR spectroscopy NMR spectra were recorded on a VarianUNITY INOVA NMR spectrometer operating at 500 MHz (1H) at 50 jC. Samples, previously sonicated to decrease the molecular weight, were exchanged three times with 99.96% deuterium oxide by lyophilisation, and finally dissolved in 99.96% deuterium oxide. Chemical shifts are expressed in ppm
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using acetone as internal reference (2.23 ppm for 1H, 31.07 ppm for 13C).
Table 3 Linkage analysis of polysaccharides Linked sugara
3. Results 3.1. Identification and typing of the isolates All isolates were classified as B. cenocepacia; six of them (three clinical and the environmental isolates) were assigned to recA lineage IIIB, whereas the remaining three isolates of clinical origin were assigned to recA lineage IIIA (Table 1). Amplification of genomic DNAs of the B. cenocepacia isolates gave rise to a total of 45 bands. Each RAPD pattern was constituted of 8 to 28 bands (mean value, 19 bands). Each isolate showed the same amplification pattern in four independent experiments (data not shown). All environmental isolates had unique RAPD fingerprints as well as the clinical isolates belonging to recA lineage IIIB (Table 1). Two of the clinical isolates (FC30 and FC42) belonging to recA lineage IIIA turned out to belong to the same RAPD type, whereas the other one (FC52) had a different RAPD fingerprint (Table 1).
2-Rha t-Gal 3-Glc 2-Hexf c 4-Glc 6-Gal 3,6-Man 4,6-Glc
Relative molar ratios RRTb
Cepacian
FC24
FC30
FC87
MDIIP144
0.80 0.88 1.00 1.02 1.09 1.14 1.27 1.28
1.00 2.88 1.02
1.00 2.62 1.08
1.00 2.97 1.12
1.00 2.36 1.12 0.12 0.20 0.25 0.81 0.03
1.00 2.90 1.06
0.43
0.43 0.27 0.54 0.06
0.61
0.26 0.67
a
The numbers indicate the position of the glycosidic linkage, t stands for non-reducing terminal. b Retention time relative to 3-linked glucose. c Unidentified 2-linked hexose in the furanose form.
its high resistance to cleavage. The sample FC52 contained the same sugars mentioned above together with Kdo. The absolute configuration of the neutral sugars was D. No information was obtained for the Kdo residue and for the glucuronic acid, due to its high resistance to methanolysis. 3.4. Linkage analysis
3.2. Exopolysaccharides production The bacterial strains were grown on solid medium containing excess of mannitol as carbon source (medium MM), previously reported to increase the production of EPS [27]. Each strain was grown on the same number of Petri dishes, and remarkably the clinical strains produced on the average more EPS than the environmental strains (404 and 290 mg, respectively). 3.3. Composition analysis of exopolysaccharides The colorimetric tests showed a content of neutral sugars between 59% and 78% and of uronic acids between 16% and 23%. The composition of the exopolysaccharides was obtained after hydrolysis of the polymers and derivatisation into alditol acetates. All the samples contained rhamnose, mannose, galactose and glucose in similar amounts. The results obtained were compared with the composition of Cepacian (Table 2). Methanolysis of the exopolysaccharides, followed by derivatisation of the methyl glycosides into trimethylsilyl ethers, gave rhamnose, mannose, galactose, glucose and glucuronic acid, the latter in low amounts, probably due to
Four samples, FC24, FC30, FC87 and MDIIP144, were subjected to linkage analysis (Table 3) and the data were compared with those described for Cepacian [8]. All the samples contained 2-linked rhamnose, terminal-galactose, 3-linked glucose and 3,6-linked mannose with molar ratios very similar to those of Cepacian. The sample FC30 contained only the sugar residues attributable to Cepacian, while the samples FC24, FC87 and MDIIP144 showed minor extraneous components. Sugar residues present in less than 10% were not considered. 3.5. 1H-NMR spectroscopy All samples were analysed by means of NMR spectroscopy. As an example, the 1H-NMR spectrum of FC42 is reported in Fig. 2. The relevant signals occur in three regions: the anomeric region (d5.6 – 4.9 for a-anomers and d4.9– 4.3 for h-anomers), the ring proton region (d4.5– 3.0), and the high field region with the methyl groups of rhamnose (f d1.3) and acetyl substituents (d2.2 – 2.0). FC42 sample exhibited one resonance for the –CH3 of rhamnose and three peaks for the acetyl groups, indicating multiple sites of substitution. Moreover, integration of the
Table 2 Molar percentages of neutral sugars in the polysaccharides samples compared with values obtained for Cepacian Sugar
Cepacian
FC 24
FC30
FC42
FC52
FC75
FC87
MVPC12
MDIIR129
MDIIP144
Rha Man Gal Glc
18 12 48 22
18 8 45 29
22 8 49 22
25 8 47 21
21 6 57 16
23 7 49 21
20 8 49 23
23 8 49 20
23 7 49 21
22 8 41 30
L. Chiarini et al. / Journal of Cystic Fibrosis 3 (2004) 165–172
Fig. 2. 1H-NMR spectrum of the sample FC42 recorded at 50 jC. The spectrum is divided in regions, as reported in the text.
methyl signals of the acetyl groups with respect to the methyl signal for the rhamnose residue showed a content of about 2.7 acetyl substituents per each rhamnose unit, indicating the presence of more then two acetyl groups per repeating unit. The anomeric region is generally a good fingerprint for the comparison of samples of unknown structure with characterised polymers. However, substitution of a ring hydroxyl function with an acetyl group causes the geminal proton signal to shift from the ring proton region to the anomeric region, thus complicating that part of the spectrum. Besides this difficulty, the 1H-NMR spectrum of all samples was very similar to that of Cepacian [8] (data not shown), apart from FC52 sample. In fact, its 1H-NMR spectrum (Fig. 3) showed resonances that were not present in the other samples. In the high field region of the spectrum, two low intensity resonances at d2.55 and 1.87 were attributed to methylene protons; the methyl protons of rhamnose gave two signals at about d1.3, and integration of the peaks at d2.20 indicated 2.4 O-acetyl groups per rhamnose residue content. Besides the main signals present in all samples examined, in the anomeric region, there were four other resonances at d5.12, 4.93, 4.77, and 4.70. These signals, together with the methylene proton peaks, are identical to those found in the 1H-NMR spectrum of the exopolysaccharide produced by the strain BTS13 [12] (Fig. 1, Formula 2). Their assignment is reported in Fig. 3. The signal at d4.93 belongs to the H-2 of the galactose residue substituted with an acetyl group on C-2. With the aim of better characterising the structure of the polysaccharides, 1H-NMR spectra were recorded on deacetylated samples that exhibited the anomeric regions of the spectra cleared of the signals due to the acetyl substitution. The anomeric regions of the 1H-NMR spectra were compared with that of Cepacian [9] (Fig. 4a) and it was evident that all the spectra contained the same resonances, sometimes together with other peaks. In Fig. 4, only part of
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the anomeric regions is shown because of the overlapping of the signals between 4.6 and 4.3 ppm with the residual HOD resonance. The signals’ assignment is reported in Fig. 4a. On the basis of the signals in the anomeric region and of their integration data (Table 4), the samples were divided in three groups. The sample FC52, characterised by signals belongings to Cepacian and to the EPS produced by B. cepacia strain BTS13, stands on its own (Fig. 4b). The samples FC24, FC75, and MDIIR129 (Fig. 4c) had integration values quite different from those of Cepacian, this feature being particularly evident for rhamnose and mannose residues. Moreover, they also had extraneous peaks in the region d5.08 – 4.80. The samples FC30, FC42, FC87, MVPC12 and MDIIP144 showed the same signals present in the 1H-NMR spectrum of Cepacian together with small extraneous peak, (Fig. 4d). Integration values of the anomeric resonances were in good agreement with those of Cepacian.
4. Discussion In this study, the production and structure of EPSs synthesised by both environmental and clinical strains of B. cenocepacia were investigated for the first time. All strains examined belonged to recA lineage IIIA or IIIB, the two world-wide spread lineages, and, except for two IIIA strains, they were genetically unrelated. It is interesting that the environmental isolates produced on average lower quantities of EPS than the clinical isolates. Although this data needs to be corroborated using a larger number of isolates, nevertheless, our preliminary findings might indicate a characteristic of increased virulence in the clinical strains. In terms of the primary structure of the polymers produced, no major differences among the nine strains examined were observed, regarding both the origin, clinical or environmental, and the recA lineage status. In fact, all
Fig. 3. 1H-NMR spectrum of the sample FC52 recorded at 50 jC. The assignment of the anomeric signals attributed to the galactan polymer is reported.
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Fig. 4. Partial anomeric regions of the 1H-NMR spectra of the deacetylated samples. (a) Cepacian, (b) FC52, (c) FC24, FC75, and MDIIR129, (d) FC30, FC42, FC87, MDIIP144, and MVPC12.
strains produced a polysaccharide identical to Cepacian (Fig. 1, Formula 1), the EPS most abundant among the Bcc isolates examined up to now. Some of these strains produced exclusively Cepacian, while others co-produced also small amounts of other polymers, as indicated by the NMR spectra and the linkage analysis data. Among the
latter, only the strain FC52, of clinical origin, produced relevant amounts of another exopolysaccharide, whose structure has already been reported in the literature [12] (Fig. 1, Formula 2). Estimation of the relative quantities of the two polysaccharides was achieved by integration of NMR signals (data not shown). The values obtained showed
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Table 4 Integration areas of the anomeric peaks Sugar residue
Exopolysaccharides samples Cep
FC24
FC30
FC42
FC75
FC87
MVPC12
MDIIR129
MDIIP144
a-GlcA a-Rha a-Gal a-Man h-Glc h-Gala
1.0 1.1 1.1 0.9 1.0 1.5
0.5 1.7 0.7 1.0 0.4 1.3
0.9 1.0 1.1 1.0 1.1 1.7
0.7 1.0 0.8 1.0 0.7 1.4
0.9 1.4 0.9 1.4 1.0 1.6
0.5 1.0 0.9 1.0 0.7 1.2
0.7 1.0 0.8 1.0 0.9 1.5
0.8 1.2 1.0 1.7 1.0 1.7
0.5 1.0 0.7 1.0 0.7 1.3
a
Integration of this signal was troublesome, due to overlapping peaks.
that the polymer different from Cepacian was present as 19% w/w. Small discrepancies in the result paragraph regarding the type and amount of sugars extraneous to Cepacian are due to the different techniques used. Considering for example the sample MDPIIP144, the alditol acetates analysis revealed a higher amount of glucose and a lower amount of galactose, while the linkage analysis indicated the presence of 6-linked galactose residue. This may be explained considering that different reactions were used in the two procedures and that components present in less than 10% in the linkage analysis were not taken into account. The NMR spectrum of MDPIIP144 showed three extraneous peaks between 5.1 and 4.9 ppm. Since NMR spectra are recorded on aqueous solutions of the samples without any previous chemical treatment, they provide reliable qualitative and quantitative data. However, 1HNMR chemical shifts cannot identify by themselves a particular sugar and this is the reason one usually needs to perform different types of experiments in order to determine the structure of polysaccharides. The polysaccharide depicted in Fig. 1, Formula 2, was already reported to be produced not only by two other clinical strains of B. cepacia [9,12], but also by Burkholderia pseudomallei [28,29], the causative agent of melioidosis, an infectious disease of humans and animals in Southeast Asia. Both Burkholderia species colonise the lungs of the hosts, posing the question of the biological role of the exopolysaccharide in the infected organ. Furthermore, in a recent work, Mack and Titball [30] showed that B. pseudomallei and B. cepacia share identical insertion sequences. These findings in these two closely related organisms point out that the transfer of genetic material between them is highly probable, with the possible emergence of pathogens with enhanced virulence [31]. The latter hypothesis is supported by evidence for bacteriophages capable of using both B. cepacia complex and B. pseudomallei as bacterial hosts [32]. In conclusion, the current knowledge shows that Bcc microrganisms produce four different exopolysaccharides (Refs. [5,7 –12,33] and this study) (Fig. 1), with the majority of strains synthesising Cepacian. The latter polymer seems to be characteristic of this bacterial complex and it has not been described to be produced by other species. The recent report by Chung et al. [5] underlines the importance of EPS in lung infections. A spontaneous variant
of the Bcc strain C1394 persisted in the lungs of an infected mouse, while the parent strain was rapidly cleared. The phenotype of the two strains was different, and among other characteristics, the variant produced more EPS than the parent one. It is interesting that they both produced the same polysaccharide, Cepacian, and that the change regarded the quantity and not the type of polymer produced. The structure –function correlation of EPSs is relevant to fully understand the biological activity of these molecules in relation to bacterial life. EPSs may exhibit two general functions: to constitute a physical barrier around bacterial cells or bacterial colonies and to possess specific chemical characteristics interfering with the host defence mechanisms. As far as the physical barrier is concerned, it should consist of a more or less loose matrix around bacterial cells. This may be achieved with rigid polysaccharidic chains that give rise to elongated macromolecules, which in turn are well suited to set up a large number of self-intermolecular connections. A study recently published by our group [11] pointed out the rigidity of the Cepacian polymer backbone that was demonstrated by means of viscosity and gyration radii measurements. Such a characteristic is explained by the presence of (1 ! 3) glycosidic bonds in the polysaccharide backbone and by the high degree of substitution of the glucuronic acid residue, which constitute a sterically hindered region. Regarding the presence of specific chemical characteristic, it is worth mentioning the frequent presence of acetyl groups on bacterial exopolisaccharides. In fact, it was reported that O-acetylation of specific hydroxyl functions on sugar residues might confer bacterial resistance to host phagocytes and complement [34], because the linkage to the O-acetyl groups renders the hydroxyl functions no longer available for binding the complement opsonins. It has to be stressed that a limited amount of Bcc strains was considered till now and an investigation of EPSs produced by the nine different Bcc genomovars might still reserve some surprises.
Acknowledgements The authors are grateful to the Italian Ministry of the Instruction University and Research (PRIN 2001) and the Regione Friuli Venezia Giulia for the financial support.
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