Profiling bacterial populations in BES streams with DNA sequencing
 
James Higgins, Christina Hohn, and Kenneth Belt

 
Historically, culturable bacteria residing in freshwater have been used as an index of water quality via "coliform" or "enterococci" testing. The bacterial genera and species comprising these categories have been identified using biochemical and physiological assays, which are relatively inexpensive but cannot provide as a great a specificity as molecular methods. We were interested in using DNA sequencing of the 16S ribosomal RNA (rRNA) gene to identify and characterize culturable bacteria from three BES stream sites: Pond Branch (POBR), McDonogh (MCDN), and Gwynns Falls- Gwynns Run (GFGR). Using a combination of inexpensive DNA extraction reagents and low-volume sequencing reactions, it is feasible to routinely obtain an average of ~ 1100-1200 nt (out of the total gene length of ~ 1650 nt) from each sample for a total per- sample cost of under $2.00. Sequences generated from each bacterial colony are subjected to analysis using an online 16S rRNA package, the Ribosomal Database 2, and a similarity matrix score of 97% or higher is deemed accurate enough to assign a species identity. Analysis of samples (n = 171) collected from June- September 2004 revealed 21 bacterial genera are present in the water, including Salmonella, Shigella, Citrobacter, and Pseudomonas. Interestingly, E. coli is prevalent in MCDN, a site associated with row crop agriculture, as well as Gwynns Run, a polluted urban stream. Our ultimate goal is to examine bacteria from each site every month over a yearlong period, generating > 550 sequences by the project's end, in an effort to determine if there are seasonal influences on bacterial species composition in the stream sites.
 

 
Keywords: bacteria, stream, E. coli, DNA