Source tracking of Escherichia coli using the 16S-23S intergenic spacer region of the rrnB ribosomal operon

Abstract

119 Escherichia coli isolates from nine different animal sources were subjected to denaturing gradient gel electrophoresis (DGGE) to determine sequence variations withing the 16S-23S intergenic spacer region (ISR) of the rrnB ribosomal operon. The ISR was analyzed to determine if E. coli isolates from various animal sources could be differentiated from each other. In E. coli, the 16S-23S ISR has been domonstrated to consist of non-essential sequences that are subject to frequent insertion or deletion events that may allow for differences between different isolates. DNA isolated from the E. coli animal sources was PCR amplified to isolate the rrnB operons. To prevent PCR amplification of all seven E. coli ribosomal operons by PCR amplification by using universal primers, sequence specific primers were utilized for the rrnB operon. An additional primer set was then used on these amplimers to prepare samples of the 16S-23S ISR for DGGE. DGGE results show the presence of 40 unique ISR sequences from all of the samples. The highest rate of unique banding patters, 60%, was observed for humans. The genetic profiles established by the PCR-DGGE method revealed a high genetic diversity for the E. coli isolates tested. There was also very little correlation between the ISR profiles created by the DGGE bands between the host sources. These findings suggest that the 16S-23S ISR may contain some host specificity, and that the high diversity of the E. coli isolates may allow for the assessment of environmental samples to distinguish similarities.