Comparative Assessment of Genotyping Methods for Epidemiologic Study of Burkholderia cepacia Genomovar III

Abstract

We analyzed a collection of 97 well-characterized Burkholderia cepacia genomovar III isolates to evaluate multiple genomic typing systems, including pulsed-field gel electrophoresis (PFGE), BOX-PCR fingerprinting and random amplified polymorphic DNA (RAPD) typing. The typeability, reproducibility, and discriminatory power of these techniques were evaluated, and the results were compared to each other and to data obtained in previous studies by using multilocus restriction typing (MLRT). All methods showed excellent typeability. PFGE with SpeI was more reproducible than RAPD and BOX-PCR fingerprinting. The discriminatory power of the methods was variable, with PFGE and RAPD typing having a higher index of discrimination than BOX-PCR fingerprinting. In general, the results obtained by PFGE, BOX-PCR fingerprinting, and MLRT were in good agreement. Our data indicate that different genomic-based methods can be used to type B. cepacia genomovar III isolates depending on the situation and the epidemiologic question being addressed. PFGE and RAPD fingerprinting are best suited to addressing small-scale studies (i.e., local epidemiology), whereas BOX-PCR fingerprinting is more appropriate for large-scale studies (i.e., global epidemiology). In this regard, BOX-PCR fingerprinting can be considered a rapid and easy alternative to MLRT.Cystic fibrosis (CF) is the most common hereditary disease in Caucasian populations. Clinical manifestations of CF result from a disturbance in electrolyte transport that primarily affects the respiratory and digestive systems. The CF lung is particularly susceptible to infection with a variety of opportunistic bacteria (9, 14), and exacerbations of chronic infection cause significant morbidity and mortality (36). Among the bacterial species capable of causing infection in CF are those belonging to the Burkholderia cepacia complex, which is currently comprised of nine closely related genomic species or genomovars (11,21,45). Recent work has demonstrated that the majority of infected CF patients harbor either B. cepacia genomovar III or Burkholderia multivorans (genomovar II) (1,25,38). Furthermore, limited data suggest that B. cepacia genomovar III (or perhaps certain specific strains within genomovar III) may be relatively more virulent than other species in this complex (3,12,19).The broad-spectrum antimicrobial resistance, absence of a vaccine, and virulence of certain strains have made prevention of B. cepacia complex infection an important goal in CF patient care (21, 22). However, much still remains unknown regarding the epidemiology of infection in CF. A number of previous studies have demonstrated transmission of B. cepacia complex strains between persons with CF (for reviews, see references 15, 16, 21, and 22). More recent studies indicate that the natural environment is also a likely reservoir for acquisition of B. cepacia complex strains (4, 24). Better risk assessment of potential sources of infection and the development of optimal infection control policies rely on a more complet...

Burkholderia latens sp. nov., Burkholderia diffusa sp. nov., Burkholderia arboris sp. nov., Burkholderia seminalis sp. nov. and Burkholderia metallica sp. nov., novel species within the Burkholderia cepacia complex

“…A dendrogram was derived from the UPGMA linkage of correlation coefficients between the BOX-PCR fingerprint patterns from Bcc isolates. Data for reference strains were available from previous studies (Chen et al, 2001;Coenye et al, 2002Coenye et al, , 2004LiPuma et al, 2002) or were unpublished (J. J. LiPuma).…”

Weissella ghanensis sp. nov., isolated from a Ghanaian cocoa fermentation

“…Isolate 225 lost viability during subsequent cultivation. To check clonality using randomly amplified polymorphic DNA (RAPD; Chen et al, 2001), DNA from each isolate was prepared by alkaline lysis (Coenye et al, 2002) and the DNA solutions were stored at 220 u C. Fingerprinting using RAPD was performed with primers RAPD-270 (59-TGCGCGCGGG-39) and RAPD-272 (59-AGCGGGCCAA-39), as described previously (Mahenthiralingam et al, 1996). Isolates 215 T and 194B were found to be non-clonal strains (data not shown).…”

Stenotrophomonas terrae sp. nov. and Stenotrophomonas humi sp. nov., two nitrate-reducing bacteria isolated from soil

“…In order to avoid studying duplicate isolates of the same strain, genotyping by random amplified polymorphic DNA PCR analysis (Coenye et al, 2002) and repetitive sequencebased PCR analysis with REP and BOX primers (Heyrman et al, 2005) were carried out. The three fingerprint methods generated identical patterns for isolates R-32746 and R-32768 T , but showed clear genetic differences for isolate R-32729 T (data not shown).…”