Graduate Thesis Or Dissertation
 

Detection of the Burkholderia cepacia complex in soil environments

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  • Burkholderia cepacia complex (Bcc) bacteria reside in soil, plant rhizospheres, and water, but the prevalence of Bcc in outdoor environments is not clear. In this study, we sampled a variety of soil and rhizosphere environments with which people may have contact: playgrounds, athletic fields, parks, hiking trails, residential yards and gardens. A total of 91 soil samples was obtained from three large U.S. cities (Philadelphia, PA, Cleveland, OH, and Portland, OR). In the first phase of the study, putative Bcc isolates were recovered on Burkholderia cepacia selective agar (BCSA) and trypan blue tetracycline medium (TBT). Isolates were sent to the Burkholderia cepacia Referral Laboratory and Repository, where they were identified using biochemical tests, growth at 32°C, and polymerase chain reaction (PCR) assays targeting both rRNA and recA gene sequences. Bcc isolates were genotyped by using RAPD, PFGE and rep-PCR. A total of 1013 bacterial isolates were examined, and 68 were identified as B. cepacia complex. The majority of these were B. pyrrocinia or genomovar VII (B. ambifaria); however, a few genomovar III isolates were also recovered. Fourteen (15%) of 91 soil samples yielded Bcc isolates. In the second phase of the study, DNA was extracted from 87 of the 91 soil samples and examined with PCR assays targeting Bcc 16S rRNA gene sequences. By using assays developed by LiPuma et al. (1999), 82% of the soil samples were positive for at least one Bcc genomovar, whereas 94% of samples were positive for at least one Bce genomovar using the Bauernfeind et al. (1999) assay system. Selected amplicons generated from four soil samples were cloned, and plasmids from multiple transformants (total=120) were screened by RFLP analysis. Among the clones evaluated from three of four soil samples, 90% or more had the "Burkholderia" RFLP pattern. In the remaining soil sample, only 9.5% of the evaluated clones displayed this profile. Sequence analysis of the 463bp 16S rRNA inserts from eight clones with the "Burkholderia" RFLP pattern indicated that all were from members of the Bcc. However, the four soil samples from which these clones were generated did not yield isolates identified as Bcc. This study indicates that the use of selective media may not be the best way to estimate the environmental prevalence of Bcc in soils. The natural populations of Bcc in soils with which people commonly have contact may be much higher than previously estimated.
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