Microbiological characterisation of Burkholderia cepacia isolates from cystic fibrosis patients: investigation of the exopolysaccharides produced

FEMS Microbiology Letters 209 (2002) 99^106

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Microbiological characterisation of Burkholderia cepacia isolates from cystic ¢brosis patients: investigation of the exopolysaccharides produced Cristina Lagatolla a , Silvia Skerlavaj b , Lucilla Dolzani a , Enrico A. Tonin a , Carlo Monti Bragadin a , Marco Bosco c , Roberto Rizzo b , Luisella Giglio d , Paola Cescutti b; b

a Dipartimento di Scienze Biomediche, Universita' di Trieste, via Fleming 22, I-34127 Trieste, Italy Dipartimento di Biochimica, Bio¢sica e Chimica delle Macromolecole, Universita' di Trieste, via L. Giorgieri 1, I-34127 Trieste, Italy c POLY-tech Scarl, Area di Ricerca, Padriciano 99, I-34012 Trieste, Italy d Centro di Fibrosi Cistica, IRCCS, Burlo Garofolo, via dell’Istria 65/1, I-34012 Trieste, Italy

Received 24 October 2001; received in revised form 17 January 2002; accepted 24 January 2002 First published online 5 March 2002

Abstract Eleven strains of Burkholderia cepacia were isolated directly from clinical specimens: 10 from sputum of cystic fibrosis patients, and one from a vaginal swab. They were biochemically identified using API20NE and confirmed by a PCR-based assay. The genomovar characterisation obtained by specific PCR amplification revealed seven strains belonging to genomovar I, three belonging to genomovar IIIA and one belonging to genomovar IV. All isolates were also typed by ribotyping and random amplification of polymorphic DNA analysis. Some of the characterised strains were examined for the ability to produce exopolysaccharides, with the aim of correlating the genomovar with the exopolysaccharide structure. The polysaccharides were analysed by means of methylation analysis and 1 H-NMR spectroscopy in order to determine structural similarities. It was shown that different strains are capable of producing chemically different polysaccharides. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. Keywords : Cystic ¢brosis; Genomovar ; Exopolysaccharide; Ribotyping; Random ampli¢cation of polymorphic DNA ; Burkholderia cepacia

1. Introduction Burkholderia cepacia was recently recognised as a real threat for cystic ¢brosis (CF) patients. In fact, this bacterial species colonises the lungs of CF patients causing a rapid and often fatal decline of their pulmonary functions [1]. The isolation and identi¢cation of B. cepacia from clinical specimens is troublesome because generally it grows very slowly and has a great phenotypic variability [2]. For these reasons, it is recommended to use selective media and molecular biology techniques for a correct identi¢cation of the strains [3]. Recently, Coenye et al. [4] established that B. cepacia should be considered a complex of at least seven closely related species (genomovars),

* Corresponding author. Tel. : +39 (040) 676 3685 ; Fax: +39 (040) 676 3691. E-mail address : [email protected] (P. Cescutti).

which can be di¡erentiated by a multiphasic taxonomic approach combining the results of di¡erent techniques, such as PAGE of whole cell proteins, DNA/DNA hybridization and fatty acid composition analysis. Although members of all seven genomovars were isolated from clinical specimens, strains of genomovar III seem to be associated with poor prognosis with respect to other members of the complex, so the identi¢cation at the genomovar level is actually considered important for the clinical management of CF patients. For this reason, more reliable methods for identifying the di¡erent genomovars, based on PCR ampli¢cation using speci¢c primers, have been developed [5]. At the same time, the interest was focussed on the characterisation of one putative virulence factor: the exopolysaccharide (EPS). Recent publications [6^8] demonstrated that clinical strains of B. cepacia produced mainly a polysaccharide constituted of a heptasaccharide repeating unit composed of the trisaccharidic backbone [3)-L-D-Glcp-

0378-1097 / 02 / $22.00 ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 5 1 1 - 6

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(1C3)-K-D-GlcpA-(1C3)-K-D-Manp-(1C]n . The glucuronic acid residue is substituted on C-2 and on C-3 with K-D-Galp and the disaccharide L-D-Galp-(1C2)-K-DRhap-, respectively. The mannose residue has a L-D-Galp residue linked on C-6. Furthermore, it was shown that some strains were also capable of producing a polymer constituted of a disaccharide repeating unit containing a 3-linked glucopyranosyl residue and a 3-linked galactopyranosyl residue substituted with a pyruvyl group in position 4,6 [9]. The aim of the present study was to characterise B. cepacia strains isolated from clinical specimens, mainly from CF patients in care at the Regional Centre for Cystic Fibrosis located in Trieste, Italy. Moreover, some of these strains were examined for their ability of producing exopolysaccharides. The sugar composition together with the glycosidic linkage determination and 1 H-NMR spectroscopy were used to determine the main structural characteristics of these polymers.

2. Materials and methods 2.1. Bacterial strains ATCC 25416T (genomovar I) was acquired from the American Type Culture Collection (Manassas, VA, USA). LMG 13010T (genomovar II), LMG 12615 (genomovar IIIA), LMG 13011 (genomovar IIIB), LMG 14294T (genomovar IV), LMG 10929T (genomovar V) and LMG 2216T (Burkholderia gladioli) were acquired from BCCM/LMG Bacteria Collection (Laboratorium voor Microbiologie, Universiteit Gent) and used to verify that the primers used for genomovar identi¢cation worked properly. Ten strains of B. cepacia and one of B. gladioli (Table 1)

were isolated directly from sputum of six cystic ¢brosis patients in care at the Regional Centre for Cystic Fibrosis in Trieste, Italy. Another clinical strain of B. cepacia was isolated in a vaginal swab from a patient in care at the University Hospital in Trieste. Clinical specimens were cultured on BCSA medium [10] at 32‡C, examined daily for growth and incubated up to 5 days before considering them negative. Isolated strains were identi¢ed as belonging to the B. cepacia complex using the commercial system API20NE (Biomerieux Vitek). More precise identi¢cation was achieved by PCR with speci¢c primers (see below). 2.2. DNA extraction Bacterial DNA was extracted as previously described [11]. Brie£y, bacteria of an overnight culture were washed once in Tris^HCl 10 mM, pH 8.0, EDTA 5 mM and then suspended in the same bu¡er. 0.5% SDS and 100 Wg ml31 of proteinase K (Sigma, St. Louis, MO, USA) were added and cells were incubated at 55‡C for 24 h. Proteins were then removed by phenol extraction and nucleic acids were recovered by ethanol precipitation. Pellets were dissolved in TE bu¡er (10 mM Tris^HCl, pH 8.0, 1 mM EDTA), treated with T1 RNase (Boehringer Mannheim, Mannheim, Germany) and quanti¢ed by visualisation on agarose gels. 2.3. Identi¢cation of the isolates by PCR Approximately 80 ng of DNA were incorporated into 50 Wl of di¡erent ampli¢cation reactions, using couples of primers speci¢c for the B. cepacia complex [12], for the di¡erent genomovars [5] and for B. gladioli [13], which can be phenotypically confused with the B. cepacia complex. The couples of primers used in this work are listed in Table 2 and the ampli¢cation reactions were run in the

Table 1 List of the isolated strains, their origin and their biochemical and molecular identi¢cation Strain

BTS1 BTS2 BTS3 BTS4 BTS5 BTS6 BTS7 BTS8 BTS10 BTS11 BTS12 BTS13

Origina Patient

Age Ib

Age IIc

A A B C D D E E G F F F

12 12 15 12 13 13 22 22 ^ 21 21 21

10 y 1 m 10 y 1 m 15 y 8 m 10 y 1 m 9y8m 9y8m 21 y 1 m 21 y 1 m ^ 17 y 8 m 17 y 8 m 17 y 8 m

y y y y y y y y

9m 9m 8m 5m 3m 3m 10 m 10 m

y9m y9m y9m

No. API20NE

Ampli¢cation with PSR1-PSL1

Identi¢cation at the genomovar level

0475577 0477577 0467577 0056557 0467577 0447577 0477573 1074556 ndd 0453577 0047577 0063577

+ + + + + + + 3 + + + +

gen IIIA gen IIIA gen I gen IV gen I gen I gen I B. gladioli gen IIIA gen I gen I gen I

a

Letters A^F indicate CF patients; letter G indicates a vaginal swab from a non-CF patient. Age I, age at the time of specimen collection; 12 y 9 m, 12 years and 9 months, etc. c Age II, age of ¢rst positive culture. d Not determined. b

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Table 2 Couples of primers used for the identi¢cation of the isolates by PCR Identi¢cation level

Primers

Length of ampli¢cation products (bp)

B. cepacia complex [12] Genomovars [5]

PSR1+PSL1 BCRG11+BCRG12 BCRBM1+BCRBM2 BCRG3A1+BCRG3A2 BCRG3B1+BCRG3B2 BCRG41+BCRG42 BCRBV1+BCRGBV2 CMG-16-1+G-16-2

209 492 714 378 781 647 378 468

B. gladioli [13]

conditions previously described [5,12,13]. Ampli¢cation products were visualized on 1.5% agarose gel electrophoresis. 2.4. Random ampli¢cation of polymorphic DNA (RAPD) typing

ilarity (PS) between the isolates. When PS was s 90% the isolates were considered ‘strictly related’, when it was between 60 and 90% the isolates were considered ‘possibly related’ and strains with a PS 6 59% were considered ‘di¡erent’. 2.7. Bacterial growth for exopolysaccharide production

80 ng of genomic DNA was ampli¢ed with the 10-base primer 208 (ACGGCCGACC) using the PCR conditions previously described [14]. Three independent extractions for each strain were examined, to verify the reproducibility of the patterns. Ampli¢cation products were run on 2% agarose gels. The patterns were compared visually and with the aid of computer analysis. 2.5. Ribotyping Approximately 1 Wg DNA of each strain was digested separately with 40 U of EcoRI and of PvuII (Roche Diagnostic, Mannheim, Germany). Restriction fragments were separated on 0.8% agarose gel, capillary blotted onto nylon membranes (Hybond-Nþ , Amersham International) and hybridized with a digoxigenin-labelled cDNA probe prepared from a mixture of Escherichia coli 16S and 23S rRNA (DIG DNA labelling kit; Roche) [15]. Detection of hybridised fragments was performed by reaction with alkaline phosphatase-conjugated anti-DIG Fab fragments followed by addition of the colorimetric substrates of alkaline phosphatase, X-phosphate and nitro blue tetrazolium chloride, in the conditions suggested by the manufacturer (Roche). The patterns were compared visually and with the aid of computer analysis. 2.6. Computer analysis The photograph of the gel of RAPD ¢ngerprinting and the membranes of ribotyping were scanned, stored as image ¢le format and processed with GelCompare II software (Applied Maths, Kortrijk, Belgium). Similarity levels were calculated by the Dice coe⁄cient [16] for each DNA typing method and the three results were combined, so that the ¢nal result was expressed as total percentage sim-

For exopolysaccharide production, bacterial strains were grown for 3 days at 30‡C on Pseudomonas Isolation Agar (PIA) (Difco) containing 20 g bacto peptone, 1.4 g MgCl2 , 10 g K2 SO4 , 0.025 g Irgasan0 , 20 ml glycerol, 13.6 g bacto agar per litre. 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 ¢nal concentration of 5%. After stirring at 4‡C for 5 h, the cells were removed by centrifugation and the EPS was precipitated from the supernatant with 4 vols. of ethanol. The EPS was then dissolved in water and puri¢ed by repeating the precipitation procedure. Finally the EPS was dialysed ¢rst against 0.1 M NaCl and then against water, the pH adjusted to neutrality and the polymer was recovered by freeze-drying. Two of the strains were also grown on a solid medium containing excess of a carbon source, previously reported to increase the production of EPS [17]. The medium contained 2 g of yeast extract, 20 g of mannitol and 15 g of bacto agar per litre and was named MM (mannitol medium). 2.8. Analytical procedures Analytical GLC was performed on a Hewlett-Packard 5890 gas chromatograph equipped with a £ame ionisation detector and an SP2330 capillary column (Supelco, 30 m), using He as the carrier gas. The following temperature programmes were used : for alditol acetates, 200^245‡C at 4‡C min31 ; for partially methylated alditol acetates, 150^245‡C at 4‡C min31 . 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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2.9. Composition analysis of EPS Hydrolysis of the EPS was carried out with 2 M tri£uoroacetic acid at 125‡C for 1 h. Alditol acetates were prepared as previously reported [8]. For the simultaneous determination of acidic and neutral sugars [8], the EPS was subjected to methanolysis (80‡C for 18 h in 1 M HCl in dry methanol) followed by reduction of the methyl ester groups with NaBD4 in ethanol. The methyl glycosides thus obtained were hydrolysed and derivatised to alditol acetates. 2.10. Linkage analysis Methylations were performed according to the modi¢ed Hakomori method using potassium methylsul¢nyl-methanide [8]. After methylation, the samples were puri¢ed on Sep-Pak C18 [8] prior to derivatisation into alditol acetates and GC-MS analysis. Molar ratio values were corrected by use of e¡ective carbon-response factors [8]. 2.11. NMR spectroscopy NMR experiments were performed on a 500-MHz Varian UNITY INOVA spectrometer. The 1 H-NMR spectra were recorded at a probe temperature of 80‡C. In order to decrease the molecular mass, sample solutions (1 g l31 ) were treated with a Branson soni¢er equipped with a miî . Each sample was subjected to ¢ve bursts crotip at 2.8 A of 1 min each, separated by 1 min intervals at 4‡C. After freeze-drying, the samples were dissolved in D2 O at a ¢nal concentration of 5 mg ml31 .

3. Results and discussion 3.1. Identi¢cation and typing of the isolates Eleven strains of putative B. cepacia were isolated from sputum of six CF patients. Apparently, four of the six patients carried two or even three strains, di¡ering both for the morphology of the colonies and for some of the biochemical tests included in the commercial identi¢cation

Fig. 1. Positive PCR ampli¢cation of the 12 isolates with the couples of primers speci¢c at the species/genomovar level. The numbers of the lanes correspond to the numbers of the isolates (e.g. lane 1 = BTS1). Lanes: 1, 2 and 10, ampli¢cation with primers BCRG11/BCRG12; 3, 5^7, 11^13, ampli¢cation with primers BCRG3A1/BCRG3A2; 4, ampli¢cation with primers BCRG41/BCRG42; 8, ampli¢cation with primers CMG-16-1/G-16-2458 ; M, 100-bp ladder (the more intense band corresponds to 800 bp).

system. The strains isolated from the di¡erent patients and their API20NE numerical pro¢les are shown in Table 1. More precise identi¢cation was achieved by PCR ampli¢cation of di¡erent regions. Ten isolates were positively ampli¢ed by the couple of primers PSR1-PSL1, targeting in a region of the 16S rDNA, speci¢c for the B. cepacia complex. All of them were submitted to ampli¢cation with the six pairs of primers speci¢c for the di¡erent genomovars and each strain gave only one positive ampli¢cation (Fig. 1). The results are summarised in Table 1: seven strains were identi¢ed as genomovar I, two as genomovar IIIA and one as genomovar IV. The strain BTS8, which was not ampli¢ed by PSR1-PSL1, was identi¢ed as B. gladioli by another species-speci¢c ampli¢cation reaction. The clinical strain of B. cepacia isolated from a vaginal swab of a woman who was not a CF patient was identi¢ed as a genomovar IIIA. The strains belonging to genomovars I and IIIA were typed by RAPD ¢ngerprinting and ribotyping (Fig. 2). The results of the di¡erent typing methods, combined and expressed as total PS (Table 3), showed that, in spite of their di¡erent phenotype, strains isolated from the same patient appeared always related. In fact, the two isolates from patient A, BTS1 and BTS2, had identical ribotype patterns and their RAPD pro¢les di¡ered only for two faint bands. Their total PS was 95.2, so we concluded that they were ‘strictly related’, although colonies of BTS2 produced a red pigment on

Table 3 Total percentage similarity between the isolates BTS5 BTS7 BTS6 BTS1 BTS2 BTS3 BTS11 BTS13 BTS12 BTS10

100 93.3 84.9 46.1 40.3 45.9 51.1 33.3 34.4 28.8

100 90.8 54.0 45.9 50.9 60.1 38.1 37.1 32.1

100 45.3 43.4 44.4 57.8 47.2 36.0 39.0

100 95.2 52.4 50.2 30.4 42.7 44.4

100 46.1 44.6 34.0 37.0 41.2

100 88.7 53.8 68.9 40.0

100 57.4 65.7 47.2

Reported values were obtained combining the Dice coe⁄cients resulting from RAPD analysis and from ribotyping.

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100 92.8 43.1

100 44.4

100

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Fig. 2. DNA ¢ngerprinting analysis of the clinical isolates by RAPD (A) and ribotyping after digestion of chromosomal DNA with EcoRI (B) and PvuII (C). The numbers of the lanes correspond to the number of the isolates (e.g. lane 1 = BTS1). Lane M, VDNA cleaved with EcoRI and HindIII (MW marker III, Roche Diagnostic, Mannheim, Germany). Lane S, mixture of VDNA cleaved with HindIII and of SPP1 DNA cleaved with EcoRI (MW marker II+MW marker VII, DIG labelled, Roche).

Luria^Bertani agar while BTS1 did not produce any kind of pigment. In the case of patient D, the strains BTS5 and BTS6 resulted quite identical from RAPD analysis and EcoRI ribotyping, while PvuII ribotyping revealed some di¡erences, resulting in a total PS of 84.9. With regard to the isolates from patient F, two of them (BTS12 and BTS13) gave a PS of 92.8, so they were considered ‘strictly related’ in spite of the mucoid aspect of BTS13 in comparison to BTS12. On the basis of the typing results and clinical history of patients A, D and F, which have been colonised by B. cepacia for more than 2 years, we hypothesised that the di¡erent isolates coming from each patient should really be considered as one strain, which eventually had undergone few mutations (evidenced by slight di¡erences in the typing pro¢les) inside the lung of the patient. Based on this assumption, we chose to investigate the EPS production of only one isolate from each patient. 3.2. Chemical characterisation of EPS Some of the characterised strains were chosen for investigating EPS production. The attention was focussed on strains isolated from di¡erent patients, and belonging ei-

ther to genomovar I, known to be more represented by environmental strains, or to genomovar IIIA. Among these, the strains which showed the most mucoid phenotype on PIA were selected. Therefore, the strains BTS2, BTS3, BTS5, BTS7, BTS10, BTS13 together with the ATCC 25416 were considered. The type of monosaccharide present in each EPS as well as the molar ratios relative to rhamnose are reported in Table 4. For comparison, the data of the EPS IST408 [8], the polysaccharide produced by a B. cepacia strain isolated in a Portuguese hospital, are included in Table 4. All the EPSs examined contained the neutral monosaccharides present in IST408 : rhamnose, mannose, galactose and glucose. Some strains showed the presence of other sugars: strains BTS2 and 13 contained arabinose, strain BTS3 contained N-acetylgalactosamine and BTS10 presented a heptose residue, which may derive from lipopolysaccharide contamination [18]. The EPSs were treated for the simultaneous detection of neutral and acidic sugars. GLC-MS analysis of the resulting mixture revealed the presence of a new component in some EPSs (Table 4): the peak corresponding to native glucose also included 6,6-dideuterium-labelled glucose, thus identifying the acidic component with glucuronic acid. Of the strains examined, it is clear

Table 4 Composition of EPSs produced by di¡erent strains of B. cepacia grown on PIA medium and sugar molar ratio relative to rhamnose

Rha Man Gal Glc Ara GalNAc Hep GlcA

IST408

BTS2

BTS3

BTS5

BTS7

BTS10

BTS13

ATCC 25416

1.0 0.7 3.9 1.4

1.0 0.5 15.0 0.5 0.6

1.0 0.5 3.3 1.2

1.0 0.6 1.5 0.7

1.0 0.5 3.0 1.4

1.0 0.3 2.0 1.0

1.0 1.4 24.4 0.6 2.5

1.0 0.9 3.0 1.4

p

p

0.3 p

nd

p

0.5 p

nd

p

Results for IST408 are reported for comparison. Rha, rhamnose ; Hep, heptose ; p, present; nd, not detected.

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35% in BTS5 and 25% in BTS10, calculated as explained before). In Fig. 3, the anomeric regions of the proton NMR spectra of EPSs IST408, BTS2 and BTS5 are depicted, together with the anomeric proton assignment for the residues belonging to the IST408 polymer. The spectrum of EPS BTS2 is completely di¡erent from that of EPS IST408, thus con¢rming the data obtained by chemical analysis, while the spectrum of EPS BTS5 clearly shows signals belonging to the same residues present in the EPS IST408, together with other signals not yet assigned, thus proving that EPS BTS5 is a mixture of two di¡erent polysaccharides. Moreover, looking at the sugar residues present in EPS BTS5, most probably the second polymer produced did not contain charged sugars. Therefore, EPS BTS5 was treated with cetyltrimethylammonium bromide [19] in order to separate the negatively charged polysaccharide from the neutral one. Hydrolysis and derivatisation to alditol acetates, followed by GLC analysis of the fraction which co-precipitated with cetyltrimethylammonium bromide gave the following molar ratio: Rha = 1.0; Man = 0.5; Gal = 3.1; Glc = 1.3. These values are very close to the one reported for EPS IST408 (Table 4), thus con¢rming the previous ¢nding. In conclusion, the data presented indicate that a fairly good number of B. cepacia clinical isolates belong to genomovar I, which is usually more represented among the environmental strains. At the same time, no direct correlation between genomovar and type of EPS produced was found. Even in the case of strains BTS5 and BTS7, which present a high degree of similarity (Table 3), the polysaccharides produced are di¡erent (Table 5). In fact BTS5 produced two di¡erent polymers, an acidic one identical to EPS BTS7, and a neutral one, whose primary structure has yet to be determined. Regarding the EPS production,

that BTS2 and BTS13 produce an EPS composed mainly of galactose, a novel polysaccharide among the ones reported in the literature up to now. The strains BTS7 and BTS13 were also grown on the MM medium and the polysaccharides produced were isolated, puri¢ed and subjected to composition analysis, which revealed the presence of the same sugar residues in the same molar ratios found in the polymers produced on the PIA medium. However, the use of mannitol as carbon source, instead of glycerol, caused strain BTS7 to produce 10 times more EPS, while it did not a¡ect the EPS production of strain BTS13 (data not shown). More precise information on the EPS primary structure was obtained by examining the glycosidic linkage pattern (Table 5). Molar ratios were calculated relative to 3-linked glucose (3-Glc) or 4-linked galactose (4-Gal). Data for the EPS IST408 are reported for comparison and the sugar residues belonging to this EPS are in bold. Monosaccharide derivatives present in less than 0.10 relative molar ratio are not shown. The strains BTS2 and BTS13 produced a polymer constituted of a trisaccharide repeating unit containing one residue of galactose linked on C-4 and two residues of galactose linked on C-3. EPS BTS7 contains only the residues characteristic of IST408 in a similar molar ratio, suggesting that the polymer produced is identical to it. Strain ATCC 25416, a phytopathogen, produced an EPS similar if not identical to IST408, together with traces (about 9%, calculated from the relative molar ratio values of the ‘non-IST408’ residues with respect to the sum of the relative molar ratios for all the sugar residues) of another polymer characterised by a trisaccharide repeating unit. Each of the strains BTS3, BTS5 and BTS10 most probably produced two types of EPSs, one identical to IST408 and the other completely di¡erent, the latter being present in lower amounts (roughly 22% in BTS3, Table 5 Determination of the position of glycosidic linkages Sugar residue

RRT

IST408

2-Rha t-Gal 3-Glc 3,6-Man 3-Rha 4-Rha t-Hex 3-Man x-Hep 3-Gal 2-Hex 4-Gal 2,3-Hex 2,6-Hex

0.91 1.00 1.14 1.44 0.92 0.93 0.93 1.15 1.15 1.18 1.22 1.23 1.35 1.46

0.98 2.82 1.00 0.42

BTS2

BTS3

BTS5

BTS7

BTS10

0.60 2.41 1.00 0.42

0.60 2.30 1.00 0.37 0.76 0.46

0.63 1.98 1.00 0.40

0.49 1.40 1.00 0.35 0.73

0.21

BTS13

ATCC 25416 0.43 1.58 1.00 0.45

0.10 0.87 1.82

1.02

0.25

0.20 0.13

1.60 0.13

1.00 0.20

1.0 0.10

The results are reported as molar ratio relative to 3-linked glucose or 4-linked galactose. The number besides each sugar residue indicates the position of the glycosidic linkage (i.e. 2-Rha = 2-linked rhamnose). RRT, relative retention time. x-Hep: the carbon atom involved in the glycosidic linkage was not determined. Monosaccharide derivatives present in less than 0.10 relative molar ratio were not reported.

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charide : C3)-Galp-(1C3)-Galp-(1C4)-Galp-(1C. This polymer is a novel one among those produced by B. cepacia and it will be investigated in detail to completely determine its primary structure, inclusive of non-carbohydrate substituents.

Acknowledgements This work was carried out with the ¢nancial support provided by the University of Trieste. The authors thank POLY-tech Scarl for the use of the Hewlett-Packard 5890 gas chromatograph and of the Hewlett-Packard 5971 mass selective detector. Dr M. Scacco is kindly acknowledged for experiments carried out during her thesis.

References

Fig. 3. Expansions of the anomeric regions of the 1 H-NMR spectra of the EPSs IST408, BTS2 and BTS5 recorded at 500 MHz.

the reported data indicated that di¡erent strains of B. cepacia can produce di¡erent EPSs: one strain (BTS7) produces exclusively one EPS, structurally identical to the one produced by a French isolate [6] and by a Portuguese isolate [8]. Other strains produce more than one EPS simultaneously, while strains BTS2 and BTS13 produce a polysaccharide containing the following repeating trisac-

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