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Journal of Clinical Microbiology, March 2008, p. 1151-1152, Vol. 46, No. 3
0095-1137/08/$08.00+0     doi:10.1128/JCM.01595-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Prevalence of Indeterminate Genetic Species of Burkholderia cepacia Complex in a Cystic Fibrosis Center in Argentina

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The Burkholderia cepacia complex (BCC) represents a group of gram-negative bacilli usually found ubiquitously in the environment whose members are of significant pathogenic potential, particularly for patients with cystic fibrosis (CF) (3). In recent years, taxonomic advances have demonstrated that this group of bacteria consists of at least nine related genetic species (formerly designated "genomovars") (9). Representative strains of all BCC species have been isolated from pulmonary infections in CF patients, but several studies have indicated that B. multivorans and B. cenocepacia account for the majority of BCC isolates from these patients (1, 6). Furthermore, certain clones of B. cenocepacia and B. dolosa are particularly associated with poor clinical course and high mortality (1, 10).

Currently, there are no data about the distribution of BCC species among the CF population in Argentina. Molecular techniques are required for an accurate identification of BCC species (7). Among these techniques, PCR-based diagnostic tests targeting the recA gene are amenable for use in the clinical laboratory (7). In our experience examining sputum samples, the BCC species were often difficult to identify using only PCR and restriction fragment length polymorphism (RFLP) analyses (7). Therefore, the aim of this study was to determine the methods needed for identification of BCC species in sputum samples from our CF patients.

Thirty putative BCC isolates were recovered on BCSA medium (4) from sputum samples of 30 CF patients attending a CF center in Buenos Aires between 2002 and 2006. Chromosomal DNA was extracted either by phenol chloroform or boiling methods (8). Phenotypic characteristics were assigned according to the method of Henry et al. (5). The genetic species status of each isolate was determined by PCR of the recA gene and RFLP with HaeIII in combination with species-specific primers as previously described (7). To further characterize the isolates, the DNA sequence of the recA gene was determined (1) and the sequences analyzed using BLAST. The recA genes of B. stabilis LMG18870 and B. cenocepacia LMG16654 were also sequenced as control strains, obtaining 100% of identity with the sequences listed under GenBank accession numbers AF143789 and AF456025, respectively.

BCC strains were confirmed as present in the 30 CF patient isolates. Sequencing 348 bp of the recA gene allowed us to identify 14 isolates, which corresponded to B. cenocepacia (3 B. cenocepacia A and 4 B. cenocepacia B isolates), B. cepacia (4 isolates), B. stabilis (2 isolates), and B. multivorans (1 isolate). However, the recA nucleotide sequences in the remaining 16 BCC isolates exhibited high-level identity with two isolates of unknown genomic species status, also called indeterminate BCC species (Table 1). Although three of these indeterminate BCC isolates had the recA-RFLP H pattern that corresponds to B. cenocepacia and also yielded amplicons with specific primers for B. cenocepacia B (Table 1), the DNA sequence of their recA gene showed 100% identity with the sequence corresponding to GenBank accession number AY228543 (available in the GenBank database) and exemplified by isolate BC14, which was previously described in a study of isolates from Brazilian CF patients (2).

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TABLE 1. Genotypic and phenotypic characteristics of indeterminate species of Burkholderia cepacia complex isolatesa

The remaining 13 BCC isolates harbored the recA sequence, with 99 to 100% identity with the sequence of the species corresponding to GenBank accession number AF456112. The recA-RFLP pattern of these BCC isolates was H or J (corresponding to B. stabilis) (Table 1), and for nine of these isolates a positive amplification product with the specific B. stabilis primers was obtained (Table 1). Moreover, the phenotypic characteristics of these isolates differed from those described for B. stabilis but resembled those of B. cenocepacia (Table 1). Indeed, most of them exhibited β hemolysis, formed a yellow-green pigment, and gave a positive result for the Burkholderia cepacia epidemic strain marker.

To identify a possible source of infection with BCC species in our hospital, air and surface samples were collected after an infected/colonized patient left the room. One hundred samples from 25 rooms were analyzed, and no evidence of BCC species was detected. Further studies are thus required to assess whether the environment around these patients represented a reservoir, especially for the indeterminate species of BCC, or whether patient-to-patient transmission had occurred.

In summary, phenotypic and genotypic tests indicated that 54% of the isolates clustered into two groups of indeterminate genetic species of BCC and that these species are prevalent among our CF patients. Molecular DNA-DNA hybridization studies should be carried out to assign the correct genetic species status of the BCC species prevalent in our CF patients (9). This molecular surveillance study also revealed that Argentina and Brazil exhibit a particular local epidemiology that should be confirmed with a wider regional study.

 
D.C. is a member of the Carrera del Investigador Científico, CONICET, Argentina.

This study was supported by grant M017 from UBACyT and by grant PICT 2003 13431 from ANPCyT, Buenos Aires, Argentina, to D.C. M.A.V. holds a Canada Research Chair in Infectious Diseases and Microbial Pathogenesis.

 
Published ahead of print on 16 January 2008.

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2. Detsika, M. G., J. E. Corkill, M. Magalhães, K. J. Glendinning, C. A. Hart, and C. Winstanley. 2003. Molecular typing of, and distribution of genetic markers among, Burkholderia cepacia complex isolates from Brazil. J. Clin. Microbiol. 41:4148-4153.[Abstract/Free Full Text]
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3. Govan, J. R. W., J. E. Hughes, and P. Vandamme. 1996. Burkholderia cepacia: Medical, taxonomic and ecological issues. J. Med. Microbiol. 45:395-407.[Abstract/Free Full Text]
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4. Henry, D. A., M. E. Campbell, J. J. LiPuma, and D. P. Speert. 1997. Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium. J. Clin. Microbiol. 35:614-619.[Abstract]
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5. Henry, D. A., E. Mahenthiralingam, P. Vandamme, T. Coenye, and D. P. Speert. 2001. Phenotypic methods for determining genomovar status of the Burkholderia cepacia complex. J. Clin. Microbiol. 39:1073-1078.[Abstract/Free Full Text]
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6. LiPuma, J. J., T. Spilker, L. H. Gill, P. W. Campbell, L. Liu, and E. Mahenthiralingam. 2001. Disproportionate distribution of Burkholderia cepacia complex species and transmissibility markers in cystic fibrosis. Am. J. Respir. Crit. Care Med. 164:92-96.[Abstract/Free Full Text]
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7. Mahenthiralingam, E., J. Bischof, S. K. Byrne, C. Radomski, J. E. Davies, Y. Av-Gay, and P. Vandamme. 2000. DNA-based diagnostic approaches for the identification of Burkholderia cepacia complex, Burkholderia vietnamiensis, Burkholderia multivorans, Burkholderia stabilis, and Burkholderia cepacia genomovars I and III. J. Clin. Microbiol. 38:3165-3171.[Abstract/Free Full Text]
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8. Seo, S. T., and K. Tsuchiya. 2004. PCR-based identification and characterization of Burkholderia cepacia complex bacteria from clinical and environmental sources. Lett. Appl. Microbiol. 39:413-419.[CrossRef][Medline]
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						Liliana Jordá-Vargas Nancy C. Castañeda Daniela Centrón* Departamento de Microbiología Inmunología y Parasitología Facultad de Medicina Universidad de Buenos Aires CABA Buenos Aires, Argentina José Degrossi Miguel D'Aquino Cátedra de Higiene y Sanidad Facultad de Farmacia y Bioquímica Universidad de Buenos Aires CABA Buenos Aires, Argentina Miguel A. Valvano Infectious Diseases Research Group Siebens-Drake Research Institute Departments of Microbiology and Immunology and Medicine University of Western Ontario London, Ontario N6A 5C1, Canada Adriana Procopio Laura Galanternik Hospital de Niños Ricardo Gutiérrez Gallo 1330 CABA Buenos Aires, Argentina
						* Phone: 54 11 5950 9500, ext. 2171, Fax: 54 11 4964 2554, E-mail: dcentron{at}gmail.com

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Journal of Clinical Microbiology, March 2008, p. 1151-1152, Vol. 46, No. 3
0095-1137/08/$08.00+0     doi:10.1128/JCM.01595-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jordá-Vargas, L. Articles by Galanternik, L. Search for Related Content PubMed PubMed Citation Articles by Jordá-Vargas, L. Articles by Galanternik, L.