DNA-Based Diagnostic Approaches for Identification of Burkholderia cepacia Complex, Burkholderia vietnamiensis, Burkholderia multivorans,Burkholderia stabilis, and Burkholderia cepacia Genomovars I and III

ABSTRACT Bacteria of the Burkholderia cepacia complex consist of five discrete genomic species, including genomovars I and III and three new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), andBurkholderia vietnamiensis (formerly genomovar V). Strains of all five genomovars are capable of causing opportunistic human infection, and microbiological identification of these closely related species is difficult. The 16S rRNA gene (16S rDNA) and recAgene of these bacteria were examined in order to develop rapid tests for genomovar identification. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA revealed sequence polymorphisms capable of identifying B. multivorans andB. vietnamiensis but insufficient to discriminate strains of B. cepacia genomovars I and III and B. stabilis. RFLP analysis of PCR-amplified recAdemonstrated sufficient nucleotide sequence variation to enable separation of strains of all five B. cepacia complex genomovars. Complete recA nucleotide sequences were obtained for 20 strains representative of the diversity of the B. cepacia complex. Construction of a recA phylogenetic tree identified six distinct clusters (recA groups):B. multivorans, B. vietnamiensis, B. stabilis, genomovar I, and the subdivision of genomovar III isolates into two recA groups, III-A and III-B. Alignment of recA sequences enabled the design of PCR primers for the specific detection of each of the six latter recA groups. The recA gene was found on the largest chromosome within the genome of B. cepacia complex strains and, in contrast to the findings of a previous study, only a single copy of the gene was present. In conclusion, analysis of the recA gene of theB. cepacia complex provides a rapid and robust nucleotide sequence-based approach to identify and classify this taxonomically complex group of opportunistic pathogens.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  P. de Vos,et al.  Identification and Population Structure ofBurkholderia stabilis sp. nov. (formerly Burkholderia cepacia Genomovar IV) , 2000, Journal of Clinical Microbiology.

[3]  P. Vandamme,et al.  Diagnostically and Experimentally Useful Panel of Strains from the Burkholderia cepacia Complex , 2000, Journal of Clinical Microbiology.

[4]  P. Vandamme,et al.  Development of rRNA-Based PCR Assays for Identification of Burkholderia cepacia Complex Isolates Recovered from Cystic Fibrosis Patients , 1999, Journal of Clinical Microbiology.

[5]  T. Heulin,et al.  Differentiation of Burkholderia Species by PCR-Restriction Fragment Length Polymorphism Analysis of the 16S rRNA Gene and Application to Cystic Fibrosis Isolates , 1999, Journal of Clinical Microbiology.

[6]  A. van Belkum,et al.  Identification of Burkholderia spp. in the Clinical Microbiology Laboratory: Comparison of Conventional and Molecular Methods , 1999, Journal of Clinical Microbiology.

[7]  J. Lipuma,et al.  Commercial use of Burkholderia cepacia. , 1999, Emerging infectious diseases.

[8]  A. Bauernfeind,et al.  Discrimination of Burkholderia gladiolifrom Other Burkholderia Species Detectable in Cystic Fibrosis Patients by PCR , 1998, Journal of Clinical Microbiology.

[9]  P. Vandamme,et al.  Agricultural and medical microbiology: a time for bridging gaps. , 1998, Microbiology.

[10]  J. Lipuma,et al.  BURKHOLDERIA CEPACIA: Management Issues and New Insights , 1998 .

[11]  P. Vandamme,et al.  Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov. , 1997, International journal of systematic bacteriology.

[12]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[13]  C. F. Gonzalez,et al.  Mobilization, cloning, and sequence determination of a plasmid-encoded polygalacturonase from a phytopathogenic Burkholderia (Pseudomonas) cepacia. , 1997, Molecular plant-microbe interactions : MPMI.

[14]  D. Simpson,et al.  Identification and characterization of a novel DNA marker associated with epidemic Burkholderia cepacia strains recovered from patients with cystic fibrosis , 1997, Journal of clinical microbiology.

[15]  J. Lipuma,et al.  Identification of Burkholderia cepacia isolates from patients with cystic fibrosis and use of a simple new selective medium , 1997, Journal of clinical microbiology.

[16]  M E Campbell,et al.  Epidemiology of Burkholderia cepacia infection in patients with cystic fibrosis: analysis by randomly amplified polymorphic DNA fingerprinting , 1996, Journal of clinical microbiology.

[17]  B. Manning,et al.  Genomic complexity and plasticity of Burkholderia cepacia. , 1996, FEMS microbiology letters.

[18]  V. Deretic,et al.  Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. , 1996, Microbiological reviews.

[19]  V. Deretic,et al.  Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia , 1996 .

[20]  P. de Vos,et al.  Polyphasic Taxonomy , a Consensus Approach to Bacterial Systematics , 1996 .

[21]  S Karlin,et al.  Bacterial classifications derived from recA protein sequence comparisons , 1995, Journal of bacteriology.

[22]  B. Tümmler,et al.  A physical genome map of the Burkholderia cepacia type strain , 1995, Molecular microbiology.

[23]  T. Heulin,et al.  Polyphasic Taxonomy in the Genus Burkholderia Leading to an Emended Description of the Genus and Proposition of Burkholderia vietnamiensis sp. nov. for N2-Fixing Isolates from Rice in Vietnam , 1995 .

[24]  M. Noble,et al.  Xanthomonas maltophilia misidentified as Pseudomonas cepacia in cultures of sputum from patients with cystic fibrosis: a diagnostic pitfall with major clinical implications. , 1995, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[25]  R. Goldstein,et al.  Cable (cbl) type II pili of cystic fibrosis-associated Burkholderia (Pseudomonas) cepacia: nucleotide sequence of the cblA major subunit pilin gene and novel morphology of the assembled appendage fibers , 1995, Journal of bacteriology.

[26]  D. Speert,et al.  Infection with Pseudomonas cepacia in chronic granulomatous disease: role of nonoxidative killing by neutrophils in host defense. , 1994, The Journal of infectious diseases.

[27]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[28]  T. Lessie,et al.  Multiple replicons constituting the genome of Pseudomonas cepacia 17616 , 1994, Journal of bacteriology.

[29]  T. Nakazawa,et al.  Cloning, sequencing, and transcriptional analysis of the recA gene of Pseudomonas cepacia. , 1990, Gene.

[30]  R. V. Miller,et al.  Construction of a recA mutant of Burkholderia (formerly Pseudomonas), cepacia , 1998, Applied Microbiology and Biotechnology.

[31]  P. Vandamme,et al.  Burkholderia (Pseudomonas) cepacia and cystic fibrosis: the epidemiology in Belgium. , 1996, Acta clinica Belgica.

[32]  P. McNabb,et al.  Pseudomonas cepacia. , 1986, Infection control : IC.