Culture-based and non-growth-dependent detection of the Burkholderia cepacia complex in soil environments

Burkholderia cepacia complex (Bee) bacteria reside in soil, plant rhizospheres, and water, but their prevalence and distribution in outdoor environments is not clear. 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 sites was sampled in three large U.S. cities. In the first phase of the study, putative Bee isolates were recovered on Burkholderia cepacia selective agar and trypan blue tetracycline medium and subsequently examined for biochemical reactivity and growth at 32 and 22degreesC. Isolates were further examined by PCR assays targeting Bcc-specific ribosomal DNA and recA gene sequences. Among the 1,013 bacterial isolates examined, 68 were identified as Bee; 14 (15%) of 91 sampled sites yielded Bee isolates. In the second phase, DNA was extracted directly from soil samples and examined with PCR assays targeting Bee 16S rRNA gene sequences. Either 82 or 93% of the soil samples were positive for at least one Bee genomovar, depending on the PCR assay system used. Cloning and sequencing were performed to check the specificity of the PCR assays. Sequence analysis of the 463-bp 16S rRNA inserts from eight clones indicated that all were from members of the Bee. The four soil samples from which these clones were generated did not yield isolates identified as Bee. Based on PCR detection, Bee appears to be prevalent in soil from urban and suburban environments. Culture-based recovery of Bee may underestimate environmental populations.