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CHARACTERIZATION OF PRECURSOR FOR 16S rRNA FOR AEROMONAS HYDROPHILA
Citation:
Revetta*, R P., J W. Santo Domingo*, D. B. Oerther, P. G. Stroot, AND B. K. Kinkle. CHARACTERIZATION OF PRECURSOR FOR 16S rRNA FOR AEROMONAS HYDROPHILA. Presented at NRMRL Peer Review, Cincinnati, OH, August 25, 2004.
Impact/Purpose:
To inform the public.
Description:
Current strategies for monitoring drinking water quality involve culture-based methods to detect the presence of microbial indicators. However, these methods are insensitive when the organisms have undergone physiological changes such as injury and starvation that can occur in hostile environments such as drinking water distribution systems. Molecular methods have been developed to address this problem but, due to the variable persistence of nucleic acids in cells post-death, may lead to false positive results. Recently, it has been suggested that changes in the levels of precursor 16S ribosomal RNA may be a sensitive indicator of physiological cell activity because of its short half-life within microbial cells. With this in mind, we initiated studies with the opportunistic human pathogen Aeromonas hydrophila ATCC 7966 to determine the presence of precursor 16S rRNA in chloramphenicol induced cells. RNA was isolated from A. hydrophila under three growth conditions, overnight (starved), stimulated with fresh media (active), and after treatment with chloramphenicol to induce precursor levels. Reverse transcription using a fluorescent-labeled universal oligonucleotide probe (Eub 338) generated a fragment of approximately 489 nucleotides. As a result, the precursor fragment size was estimated to be 150 nucleotides in length. Based on published data for the 16S-23S rRNA intergenic spacer region of A. hydrophila ATCC 7966, primers were developed for the precursor region upstream of the mature rRNA molecule. When the upstream primers were used in combination with primer Eub 8, PCR products of the expected size were obtained, and subsequently sequenced. A search of sequence databases suggests this may be a good target region for the development of species-specific primers for real time quantitative analysis of A. hydrophila cells exposed to different disinfection and starvation treatments.