Molecular characterization of Burkholderia cepacia isolates from cystic fibrosis (CF) patients in an Italian CF center

Molecular characterization of Burkholderia cepacia isolates from cystic fibrosis (CF) patients in an Italian CF center

Research in Microbiology 154 (2003) 491–498 www.elsevier.com/locate/resmic Molecular characterization of Burkholderia cepacia isolates from cystic fi...

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Research in Microbiology 154 (2003) 491–498 www.elsevier.com/locate/resmic

Molecular characterization of Burkholderia cepacia isolates from cystic fibrosis (CF) patients in an Italian CF center Andrea Petrucca a , Paola Cipriani b , Piera Valenti c , Daniela Santapaola d , Carmen Cimmino e , Gian Luca Scoarughi e , Iolanda Santino b , Stefania Stefani f , Rosa Sessa b , Mauro Nicoletti d,∗ a Azienda Ospedaliera “Sant’Andrea”, Laboratorio di Microbiologia Clinica, Via di Grottarossa 1035, 00189 Rome, Italy b Dipartimento di Scienze di Sanità Pubblica, Università “La Sapienza”, P. le Aldo Moro 5, 00185 Rome, Italy c Dipartimento di Medicina Sperimentale, II Università di Napoli, 80135 Naples, Italy d Dipartimento di Scienze Biomediche, Università “G. D’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy e Dipartimento di Biologia Cellulare e dello Sviluppo, Sezione di Scienze Microbiologiche, Via dei Sardi 70, 00185 Rome, Italy f Dipartimento di Scienze Microbiologiche e Ginecologiche, Università di Catania, Via Androne 81, 95124 Catania, Italy

Received 2 July 2002; accepted 24 June 2003

Abstract Bacteria of the Burkholderia cepacia complex consist of a number of closely related genomic species (genomovars) potentially pathogenic for cystic fibrosis (CF) patients, collectively referred to as the B. cepacia complex. The genomovar status and epidemiological relatedness of B. cepacia complex strains recovered from CF patients, attending a CF Center at the University Hospital “Policlinico Umberto I” of Rome, were investigated using 16S rRNA PCR-RFLP, recA PCR-RFLP, genomovar-specific PCR, and RAPD. Forty-seven isolates identified as B. cepacia by commercial systems were repeatedly recovered from 19 CF patients. The taxonomy approach used in this study showed that 17 of the 19 patients were colonized by B. cepacia complex strains. Genomovar III (11 strains) was the most prevalent genomovar. Two strains of genomovar I, one B. stabilis (genomovar IV), one B. multivorans (genomovar II), and 4 strains of B. anthina (genomovar VIII) were also identified. This is the first report of multiple patient colonization by B. anthina in a CF center. The epidemiological and genetic relatedness as well as the presence of molecular markers associated with virulence and transmissibility of the B. cepacia complex strains were determined and probable patient-to-patient spread was observed.  2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: B. cepacia; Cystic fibrosis; Genomovars; Taxonomy

1. Introduction The Gram-negative bacillus Burkholderia cepacia has been recognized as a major pathogen for cystic fibrosis (CF) patients [11]. The concern regarding the emergence of this opportunistic pathogen among CF patients is due to its increased isolation since the late 1970s, the capacity for spread of the infection among the CF patient community, its role in damaging lung functions, inducing necrotizing pneumonia and frequently fatal septicemia, and its innate multiantibiotic resistance profile [1,2,8,13,21]. The complex taxonomy and laboratory problems concerning bacterial identification (these microorganisms are often confused with * Corresponding author.

E-mail address: [email protected] (M. Nicoletti).

the so-called “B. cepacia-like” microorganisms) have led to the development of phenotypic and molecular (PCR-based) methods to correctly identify these bacteria [10,15,19,24, 25,29–31]. Molecular methods are based on nucleotide sequence polymorphism of the 16S rRNA and of the recA genes [10,15,19,24,29]. A rapid and precise identification of bacteria belonging to the “B. cepacia complex” is of crucial importance to fully define specific risks, to influence segregation of CF patients and infection control strategies [7,12]. Recent research has shed some light on the taxonomy of the B. cepacia, and the name “B. cepacia complex” was proposed to comprise a cluster of nine related species (or genomovars) associated with respiratory tract infections of CF patients [5,29]. In this study we have characterized at a molecular level 47 clinical isolates, presumptively identified as B. cepacia by the commercial API 20NE system, isolated from sputa of

0923-2508/$ – see front matter  2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0923-2508(03)00145-1

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19 CF patients attending a CF Center at the University Hospital “Policlinico Umberto I” of Rome. The epidemiological and genetic relatedness as well as the presence of molecular markers believed to be associated with virulence and transmissibility of these isolates were also determined.

fingerprints was calculated with the Dice coefficient. A 1.2% tolerance in fragment migration was applied for comparison of the fingerprints. Clustering analysis was performed with the unweighted pair group method with arithmetic average (UPGMA).

2. Materials and methods

2.3. Restriction fragment length polymorphism (RFLP) analysis of amplified 16S rDNA

2.1. Bacterial strains and growth conditions CF patients, registered at the CF Center of the University Hospital “Policlinico Umberto I” of the University “La Sapienza” of Rome between 1995 and 2000, were screened for the carriage of B. cepacia from sputum plated on “B. cepacia medium” (MAST medium, Mast Diagnostics, UK) supplemented with Selectatab. Media were incubated at 35 ◦ C for three days. Forty-seven isolates, obtained from 19 CF patients, were identified as B. cepacia by the API 20NE (bioMérieux) system and were molecularly characterized by several molecular methods (see below). Previously characterized B. cepacia clinical isolates of different genomovar (two B. cepacia genomovar III strains (one carrying cblA, and the other BCESM), one B. stabilis (genonomar IV) and one B. multivorans (genomovar II)) [1] were used as control strains.

Identification of B. cepacia complex isolates was accomplished by PCR-RFLP assays using the primers pairs UNI2 and UNI5 (Table 1) which amplify a 1-kb fragment internal to the 16S rRNA gene. PCR reactions were carried out in a final volume of 25 µl. The amplified 1-kb DNA fragments were digested with DdeI restriction endonuclease, as described by Mahenthiralingam et al. [15], and RFLP patterns were detected by electrophoresis on 3% agarose gels (ultrapure agarose, Life Technologies/Invitrogen) and staining with ethidium bromide. Inclusion in the B. cepacia complex group was achieved by comparing the different RFLP profiles of our isolates with those of two clinical isolates of B. cepacia genomovar III (one carrying cblA, and the other BCESM genetic markers), of B. stabilis (genonomar IV) and of B. multivorans (genomovar II) (control strains) [1] and with published 16S rRNA RFLP types reported by Mahenthiralingam et al. [15] and by Henry et al. [10] for B. cepacia complex microorganisms.

2.2. RAPD analysis 2.4. recA-based analysis Random-amplified polymorphic DNA (RAPD) analysis was conducted as described by Mahenthiralingam et al. [16,17], with some modifications. Briefly, genomic DNA was extracted from overnight bacterial cultures of clinical isolates with the aid of a DNA purification kit (Qiagen genomic-tip 20/G, Diagen), as recommended by the manufacturer. DNA concentrations were estimated spectrophotometrically (A260 ) and the quality of individual preparations was assessed by visual inspection after agarose gel electrophoresis. PCR mixtures (25 µl final volume) contained 100 ng of genomic DNA as template, 1.6 µM of primer oligonucleotide 270 (Table 1), 3 mM MgCl2 , 250 µM of each deoxynucleotide triphosphate, 1 U of Taq DNA polymerase (Life Technologies/Invitrogen) in 1× PCR buffer. PCR reaction mixtures were subjected to 34 thermal cycles according to the following scheme: 4 cycles, with 1 cycle consisting of 2.5 min at 94 ◦ C, 2.5 min at 36 ◦ C, and 2.5 min at 72 ◦ C; and 30 cycles, with 1 cycle consisting of 30 s at 94 ◦ C, 30 s at 36 ◦ C, and 1 min at 72 ◦ C. PCR amplifications (regarding the RAPD analysis as well as other molecular tests conducted in this study), were performed in a 9700 Perkin–Elmer PCR apparatus. Amplified DNA fragments were separated by gel electrophoresis in 1.5% agarose gels and stained with ethidium bromide. RAPD fingerprints (including DNA fragments comprised in the range of 0.15 and 4 kb), were analyzed by the aid of GelCompar software version 3.1 (Applied Maths, Kortrijk, Belgium). The degree of similarity among different

Determination of the genomovar status of our isolates was achieved by a combination of two different molecular methods, namely PCR-RFLP of the recA gene (PCR amplification using primers BCR1 and BCR2 (Table 1), followed by HaeIII digestion of the amplified DNA fragments), and genomovar-specific PCR of the recA gene. All amplifications were carried out as described by Mahenthiralingam et al. [15], in a final volume of 25 µl. B. cepacia genomovar identification was achieved by comparing the different recA– HaeIII RFLP profiles of our isolates, determined by agarose gel electrophoresis, with those produced, in the same experimental conditions, by the B. cepacia complex strains of known genomovar described above (control strains) [1] and with the recA-RFLP types reported by Mahenthiralingam et al. [15], Henry et al. [10] and Vandamme et al. [29]. Confirmation of B. cepacia genomovar identification was achieved by genomovar-specific PCR of the recA gene. The different genomovar-specific primer pairs, used in this assay, are designed to amplify DNA fragments specific for each of the B. cepacia genomovars listed in Table 1 [4,15,29]. 2.5. PCR amplification of the cblA and BCESM genes Oligonucleotide primers used for the detection of the epidemic markers cblA and BCESM in the studied clinical isolates are shown in Table 1 [3]. Fifty nanograms of genomic

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Table 1 Primers used in this study Specificity and primer name PCR-RFLP UNI2 UNI5 BCR1 BCR2 recA genomovar-specific PCR BCRG11 BCRG12 BCRBM1 BCRBM2 BCRG3A1 BCRG3A2 BCRG3B1 BCRG3B2 BCRG41 BCRG42 BCRGV1 BCRGV2 BCRGC1 BCRGC2 BCRG81 BCRG82 Epidemic markers CBL1 CBL2 BCESM1 BCESM2 RAPD P270

Sequence

Target

Reference

5 -GACTCCTACGGGAGGCAGCAG-3 5 -CTGATCCGCGATTACTAGCGATTC-3 5 -TGACCGCCGAGAAGAGCAA-3 5 -CTCTTCTTCGTCCATCGCCTC-3

16S rRNA

[15] [15] [15] [15]

5 -CAGGTCGTCTCCACGGGT-3 5 -CACGCCGATCTTCATACGA-3 5 -CGGCGTCAACGTGCCGGAT-3 5 -TCCATCGCCTCGGCTTCGT-3 5 -GCTCGACGTTCAATATGCC-3 5 -TCGAGACGCACCGACGAG-3 5 -GCTGCAAGTCATCGCTGAA-3 5 -TACGCCATCGGGCATGCT-3 5 -ACCGGCGAGCAGGCGCTT-3 5 -ACGCCATCGGGCATGGCA-3 5 -GCTGCAAGTCATCGCTGAA-3 5 -TACGCCATCGGGCATGCT-3 5 -GTCGGGTAAAACCACGCTG-3 5 -TCCGCAGCCGCACCTTCA-3 5 -TACGGTCCGGAATCGTCG-3 5 -CGCACCGACGCATAGAAT-3

B. cepacia (I)

5 -CCAAAGGACTAACCCA-3 5 -AGCCGATGTCCATCACA-3 5 -CCACGGACGTGACTAACA-3 5 -CGTCCATCCGAACACGAT-3 5 -TGCGCGCGGG-3

DNA of each isolate were used as template for PCR amplification reactions. PCR mixtures (25 µl final volume) also contained 2 µM of each primer oligonucleotide, 1.5 mM of MgCl2 , 200 µM of each deoxynucleotide triphosphate, 1.5 U of Taq DNA polymerase (Life Technologies/Invitrogen), in 1× PCR buffer. Cycling conditions were as those described by Clode et al. [3]. The two B. cepacia genomovar III, one positive for the carriage of cblA and the other for BCESM, described above [1] were included as controls. Amplified DNA fragments were separated by electrophoresis on 1% agarose gels and visualized by staining with ethidium bromide.

3. Results 3.1. Identification of B. cepacia complex strains based on 16S rRNA gene polymorphism RFLP analysis of a 1000-bp portion of the 16S rRNA gene was performed to identify isolates belonging to the B. cepacia complex group [15,24], among 47 putative B. cepacia clinical isolates obtained from 19 CF patients registered at the CF Center of the University Hospital “Policlinico Umberto I”. DNA fragments were digested with re-

recA

B. multivorans (II) B. cepacia (III-A) B. cepacia (III-B) B. stabilis (IV) B. vietnamiensis (V) B. ambifaria (VII) B. anthina (VIII)

cblA BCESM

[15] [15] [15] [15] [15] [15] [15] [15] [15] [15] [15] [15] [4] [4] [29] [29] [3] [3] [3] [3] [17]

striction endonuclease DdeI, which is known to produce the most discriminatory RFLP profiles for bacteria belonging to the B. cepacia complex [15]. Identification of B. cepacia complex strains was accomplished by comparison of the 16S rRNA RFLP profiles of our isolates with those reported by Henry et al. [10] and Mahenthiralingam et al. [15] and by direct comparison with those of four strains previously characterized as B. cepacia genomovar III (two strains), B. stabilis, and B. multivorans [1] (data not shown). As the different isolates recovered from each individual patient were always identical with respect to 16S rRNA gene polymorphism (this paragraph) and also to the other molecular tests used in this study (see below), we decided to present our results referring to 19 (one from each patient) rather than to the 47 clinical isolates. Fig. 1A shows the four different 16S rRNA gene RFLP profiles, named patterns 1–4, found among the clinical isolates examined. Pattern 1 (characteristic of B. cepacia genomovars I and III, B. stabilis (genomovar IV) and B. ambifaria (genomovar VII) strains) [10,15] was shared by strains that colonized 16 out of the 19 patients examined (Table 2). Of the remaining 3 patients, one was colonized by a strain showing pattern 3 typical of B. gladioli, one was colonized by an isolate presenting an RFLP profile 2 typical of B. multivorans (genomovar II) strains, and one

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(A)

(B)

Fig. 1. RFLP analysis. (A) PCR-RFLP of 16S rRNA gene performed with DdeI restriction endonuclease. Lanes 1–4 show the different RFLP patterns (from 1 to 4) found among the 47 clinical isolates examined in this study. Pattern 1 is shared by strains of B. cepacia genomovars I and III, B. stabilis (genomovar IV) and B. anthina (genomovar VIII); pattern 2 identifies B. multivorans (genomovar II); pattern 3 identifies B. gladioli; and pattern 4 corresponds to a profile incompatible with B. cepacia complex strains ([10,15] and Table 2). (B) PCR-RFLP of recA gene performed with HaeIII restriction endonuclease. Lanes 1–6 show the different RFLP patterns found among the 43 B. cepacia complex isolates identified with the PCR-RFLP of the 16S rRNA. recA–HaeIII RFLP types are indicated below each lane. Lanes M (A and B) indicate 1-kb Ladder Mix (MBI Fermentas) DNA size standard.

Table 2 Molecular identification, genomovar status and presence of the cblA and BCESM epidemic markers among representative B. cepacia complex strains isolated from 19 patients studied Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomovar-specific PCR of recA genec

16S rDNA

recA–HaeIII

DdeI-RFLP patterna

based RFLP

I

II

III-A

III-B

IV

VIII

1 1 3 1 2 1 1 1 1 1 1 1 1 1 1 1 1 4 1

G G nab J F G G E T T T E G H T G H na H

– – – – – – – + – – – + – – – – – – –

– – – – + – – – – – – – – – – – – – –

+ + – – – + + – – – – – + – – + – – –

– – – – – – – – – – – – – + – – + – +

– – – + – – – – – – – – – – – – – – –

– – – – – – – – + + + – – – + – – – –

Final identification III-A III-A B. gladioli IV (B. stabilis) II (B. multivorans) III-A III-A I VIII (B. anthina) VIII (B. anthina) VIII (B. anthina) I III-A III-B VIII (B. anthina) III-A III-B Non-B. cepacia complex III-B

Presence of:

RAPD

cblA

BCESM

typed

– – – – – – + – – – – – – – – – – – –

+ + – – – + – – – – – – + – – + – – –

1 U nte U U 1 U 2 4 U 4 2 1 3 U 1 3 nt 3

a Pattern 1 includes B. cepacia genomovars I and III, B. stabilis (genomovar IV) and B. anthina (genomovar VIII); pattern 2 corresponds to B. multivorans (genomovar II); pattern 3 corresponds to B. gladioli [10,15]; pattern 4 corresponds to a non-B. cepacia complex isolate; b na, not amplified; c I, genomovar I; II, B. multivorans (genomovar II); III-A and B, genomovar III, recA group III-A and III-B, respectively; IV, B. stabilis (genomovar IV); VIII, B. anthina (genomovar VIII); all clinical isolates were negative for both B. vietnamiensis (genomovar V) and B. ambifaria (genomovar VII); d U, unique RAPD type; 1 to 4 correspond to cluster 1 to cluster 4, respectively (Fig. 2B); e nt, not typed.

an uncleaved pattern, which corresponds to a profile not characteristic either of B. cepacia complex strains or of other closely related species [10,15] (Table 2). As expected,

control strains of B. cepacia genomovar III and B. stabilis presented a 16S rRNA gene RFLP profile of pattern 1, while B. multivorans showed a pattern 2 (data not shown).

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(A)

(B) Fig. 2. (A) RAPD analysis. Lanes 1–17, RAPD fingerprints profiles of 17 B. cepacia complex strains representative of the 43 strains examined in this study. Patient identification numbers are shown below each lane. Lanes M, 1-kb Ladder Mix (MBI Fermentas) DNA size standard. (B) Dendogram showing the results of clustering analysis using UPGMA and Dice coefficent for the 17 RAPD fingerprints profiles shown above. Scale bar (shown on the top left) indicates the percentage of genetic similarity, while the vertical dotted line represents the Dice cut-off value (70%) used to group similar RAPD profiles. Electronically reproduced RAPD profiles and molecular sizes (in kb) (arranged in a logarithmic scale) are shown on the right. The four rectangles indicates identical fingerprints (clusters). Patient identification numbers are shown on the right.

These results led us to conclude that of the 19 patients considered to be colonized by B. cepacia by presumptive identification performed by standard microbiological techniques, 17 were colonized by B. cepacia complex strains, one by B. gladioli and one by a not-fully characterized strain (patient 18).

3.2. B. cepacia genomovar identification by recA-RFLP and genomovar-specific PCR The PCR of the recA gene is a useful assay which specifically discriminates between the B. cepacia complex and “B. cepacia-like” microorganisms [19]. Using B. cepacia

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complex-specific primers BCR1 and BCR2 (Table 1) [15], a 1-kb fragment was successfully amplified from strains isolated from 17 (43 isolates) out of the 19 patients examined. As expected, the strains that did not allow amplification with these primers were those identified as B. gladioli and as nonB. cepacia strains (data not shown) (Table 2). The amplified 1-kb recA DNA fragments were digested with HaeIII restriction endonuclease. Fig. 1B shows six distinct recA–HaeIII RFLP profiles (RFLP types E, G, H, F, J and T) found among the isolates shown to belong to the B. cepacia complex. All of these were found to correspond to those described by Mahenthiralingam et al. [15], McDowell et al. [19] and Vandamme et al. [29]. Control strains of B. cepacia genomovar III presented RFLP types G and H, B. multivorans F, and B. stabilis J. These results allowed us to establish that nine patients (No. 1, 2, 6, 7, 13, 14, 16, 17 and 19) were colonized by B. cepacia genomovar III strains (six patients by recA– HaeIII RFLP type G and three by recA–HaeIII RFLP type H); one (No. 5) was colonized by B. multivorans (genomovar II, recA–HaeIII RFLP type F); and one by a strain of recA– HaeIII RFLP type J, an RFLP type shared by both B. cepacia PC184 (genomovar III) and B. stabilis (genomovar IV) (Table 2) [15]. Two patients (No. 8 and 12) were presumably colonized by strains of genomovar I (recA–HaeIII RFLP type E), and four (No. 9, 10, 11 and 15) by B. anthina (recA– HaeIII RFLP profile T), a novel genomovar of B. cepacia complex [29] (Fig. 1B and Table 2). Furthermore, to solve these ambiguities and to definitively assess the genomovar status of our isolates, total DNA of the studied and control strains was subjected to genomovar-specific PCR of the recA gene [4,15,29], using the eight previously described genomovar-specific oligonucleotide primers pairs reported in Table 1. The results obtained (not shown) confirmed our identifications and allowed to unambiguously identify the RFLP type J isolate as B. stabilis, the RFLP type T isolates as B. anthina (Table 2), and to establish that of the nine B. cepacia genomovar III isolates, six were of the recA group III-A, and three of the recA group III-B. As expected no isolate was positive when amplification was conducted with primers that are specific for B. vietnamiensis (genomovar V) or B. ambifaria (genomovar VII). 3.3. Epidemiological relatedness of the B. cepacia complex isolates All B. cepacia complex strains isolated were typed by a RAPD fingerprint assay to determine their epidemiological relatedness [16,17]. The RAPD polymorphisms of 17 B. cepacia complex strains, each representative of each individual patient, were generated with primer 270 (Table 1) and are shown in Fig. 2A. Six unique fingerprints (presenting a Dice coefficient of similarity less than 70%) and four groups of identical strains presenting profiles with a coefficient of 100% were found (Fig. 2B). Of the six unique fingerprints, two were found to belong to B. cepacia genomovar III (patients No. 2 and 7; recA group III-A), one to B. stabilis (pa-

tients No. 4; genomovar IV), one to B. multivorans (patients No. 5; genomovar II), and the remaining two to B. anthina (patients No. 10 and 15; genomovar VIII). Cluster 1 is composed of four strains of genomovar III (recA group III-A); cluster 3 of three of genomovar III (recA group III-B), cluster 2 of two strains of genomovar I; and cluster 4 of two strains of B. anthina (Table 2). 3.4. PCR detection of the cblA and BCESM genes The potential pathogenicity of the B. cepacia complex strains identified in this study was assessed determining the presence, by PCR, of cblA and of BCESM, two genetic markers believed to be associated with virulence and the capacity of B. cepacia complex microorganisms to spread among CF patients [1,3,28]. Results are shown in Table 2. B. cepacia complex strains which belong to genomovar III (recA group III-A) were found to be positive for the presence of the genetic markers, including strains isolated from five patients (No. 1, 2, 6, 13 and 16) found positive for the BCESM gene, and one (No. 7) found positive for the cblA gene (Table 2). No other B. cepacia strain was positive for any of those tests. Interestingly, of the five recA group III-A BCESMpositive strains, four were grouped in the RAPD cluster 1 (patients No. 1, 6, 13 and 16) and one showed a unique RAPD profile (patient No. 2) (Fig. 2B and Table 2). The recA group IIIA cblA-positive strain showed a unique RAPD profile (patient No. 7) (Fig. 2B and Table 2).

4. Discussion B. cepacia complex strains have been increasingly isolated worldwide from CF patients since the late 1970s [8,21]. The concern regarding the emergence of this opportunistic pathogen is principally due to its impact on both morbidity and mortality of infected CF patients, to its innate multiantibiotic resistance profile and to its epidemic spread [8,13]. Due to their taxonomic complexity and peculiar genomic characteristics, identification of B. cepacia complex isolates is a rather difficult task and requires molecular procedures mainly based on nucleotide sequence polymorphism of the 16S rRNA and of the recA genes [5,10,15,19,22,24,27,29]. In this paper, a polyphasic molecular study was performed to characterize 47 clinical isolates, isolated from sputa of 19 CF patients attending a CF Center at the University Hospital “Policlinico Umberto I” of Rome, preliminarily identified as B. cepacia by the API 20NE (bioMérieux) system. The results of our study indicate that 17 patients were colonized by strains of the B. cepacia complex group and that genomovar III was the most prevalent genomovar (nine patients, Table 2). Our results are in agreement with those of Agodi et al. [1] who recently reported B. cepacia genomovar III as the prevalent B. cepacia complex species

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among CF patients in four Italian CF Centers. Of the remaining eight patients, two were found to be colonized by B. cepacia strains of genomovar I, one by B. stabilis (genomovar IV), one by B. multivorans (genomovar II), and four patients by B. anthina (Table 2) [15,19,29]. The finding that four out of the 19 patients examined were colonized by B. anthina was a rather unexpected result, since B. anthina is commonly isolated from the rhizosphere [29] and colonization of CF patients by these isolates has been reported to occur at a very low frequency in an extensive epidemiological study regarding the molecular characterization of a considerably high number of B. cepacia complex strains isolated from the sputa of CF patients [14]. Moreover, in a similar study, Bevivino et al. [2] failed to find B. anthina as colonizing microorganisms in a large Italian CF Center as well as from environmental sources. Although our results are definitely influenced by the limited number of B. cepacia complex isolates considered in this study, the finding of four B. anthina isolates and the fact that two of these can be grouped into a cluster (cluster 4, Fig. 2B), suggesting epidemiological relatedness, indicates that the effective impact of this genomovar, in terms of pathogenicity and transmissibility among CF patients, needs to be further elucidated. Patient-to-patient transmission of B. cepacia complex strains is a well documented phenomenon and the existence of epidemic strains has also been reported among the different CF communities [13,20,26]. Conversely, the source of infection for CF patients, the mechanism(s) of virulence and the different levels of transmissibility presented by B. cepacia complex strains of different genomovars are still open questions that will necessitate further studies [2,9,14]. To assess epidemiological relatedness among B. cepacia complex isolates we conducted RAPD analysis. Of the 17 patients found to be colonized by B. cepacia complex strains, six were colonized by strains presenting a unique RAPD fingerprint (Fig. 2B and Table 2). This indicated that these patients have probably been colonized by epidemiologically unrelated strains rather than by patient to patient transmission [1,27]. On the other hand, the RAPD analysis (Fig. 2B and Table 2) also revealed that the remaining eleven isolates can be grouped into four clusters of epidemiologically related strains (the composition of each cluster was also confirmed by pulsed field gel electrophoresis (PFGE) experiments; data not shown), probably indicating patient-to-patient transmission of B. cepacia complex strains among the CF community. Moreover, chronic colonization of the patients with the same strain was also observed since multiple isolates recovered at different times from individual patients always presented identical RAPD profiles (data not shown). The presence of BCESM and of cblA, two genetic markers suspected to be associated with virulence and transmissibility of B. cepacia complex strains [1,6,18,23], was also investigated. The results obtained indicated that the two markers were present only in isolates belonging to strains of genomovar III, recA group III-A (five patients carried isolates found positive for BCESM, and one for cblA,

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Table 2). Interestingly, four of the five BCESM-positive strains are part of the RAPD cluster 1 and the three isolates of B. cepacia complex strains of genomovar III, recA group III-B are part of the RAPD cluster 3 confirming the already proposed fact that colonization of CF patients by strains of genomovar III might increase the risks of transmission of the infection to other components of the CF communities [1]. Acknowledgements We thank Mrs. Elena Nebuloso for her excellent technical assistance. This work was supported by Faculty 60% funds granted to R.S., and in part by THE MURST PRIN research project “Effettori di virulenza in patogeni enterici: caratteristiche e studio delle loro interazioni” granted to M.N. References [1] A. Agodi, E. Mahenthiralingam, M. Barchitta, V. Gianninò, A. Sciacca, S. Stefani, Burkholderia cepacia complex infection in Italian patient with cystic fibrosis: Prevalence, epidemiology, and genomovars status, J. Clin. Microbiol. 39 (2001) 2891–2896. [2] A. Bevivino, C. Dalmastri, S. Tabacchini, L. Chiarini, M.L. Belli, S. Piana, A. Materazzo, P. Vandamme, G. Manno, Burkholderia cepacia complex bacteria from clinical and environmental sources in Italy: Genomovar status and distribution of traits related to virulence and transmissibility, J. Clin. Microbiol. 40 (2002) 846–851. [3] F.E. Clode, M.E. Kaufmann, H. Malnick, T.L. Pitt, Distribution of genes encoding putative transmissibility factors among epidemic and non epidemic strains of Burkholderia cepacia from cystic fibrosis patients in the United Kingdom, J. Clin. Microbiol. 38 (2000) 1763– 1766. [4] T. Coenye, E. Mahenthiralingam, D. Henry, J.J. LiPuma, S. Laevens, M. Gillis, D.P. Speert, P. Vandamme, Burkholderia ambifaria sp. nov., a novel member of the Burkholderia cepacia complex including biocontrol and cystic fibrosis-related isolates, Int. J. Syst. Evol. Microbiol. 51 (2001) 1481–1490. [5] T. Coenye, P. Vandamme, J.R.W. Govan, J.J. LiPuma, Taxonomy and identification of the Burkholderia cepacia complex, J. Clin. Microbiol. 39 (2001) 3427–3436. [6] R. Goldstein, L. Sun, U. Jiang, J. Forstner, C. Campanelli, Structurally variant classes of pilus appendage fibers coexpressed from Burkholderia (Pseudomonas) cepacia, J. Bacteriol. 177 (1995) 1039–1052. [7] J.R.W. Govan, V. Deretic, Microbial pathogenesis in cystic fibrosis: Mucoid Pseudomonas aeruginosa and Burkholderia cepacia, Microbiol. Rev. 60 (1996) 539–574. [8] J.R.W. Govan, J.E. Hughes, P. Vandamme, Burkholderia cepacia: Medical, taxonomy and ecological issues, J. Med. Microbiol. 45 (1996) 395–407. [9] D.G. Heath, K. Hohneker, C. Carriker, K. Smith, J. Routh, J.J. LiPuma, R.M. Aris, D. Weber, P.H. Gilligan, Six-year molecular analysis of Burkholderia cepacia complex isolates among cystic fibrosis patients at a referral center for lung transplantation, J. Clin. Microbiol. 40 (2002) 1188–1193. [10] D.A. Henry, E. Mahenthiralingam, P. Vandamme, T. Coenye, D.P. Speert, Phenotypic methods for determining genomovar status of the Burkholderia cepacia complex, J. Clin. Microbiol. 39 (2001) 1073–1078. [11] A. Isles, I. Maclusky, M. Corey, R. Gold, C. Prober, P. Fleming, H. Levinson, Pseudomonas cepacia infection in cystic fibrosis: An emerging problem, J. Pediatr. 104 (1984) 206–210. [12] J.J. LiPuma, Burkholderia cepacia: Management issues and new insights, Clin. Chest Med. 19 (1998) 473–486.

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