DNA polynucleotide probes generated from representatives of the genus Acinetobacter and their application in fluorescence in situ hybridization of environmental samples

The application of rRNA directed polynucleotide probes carrying multiple la bels facilitates the detection of target cells by fluorescence in situ hybr idizations and allows specific enrichment by cell fishing. So far, exclusiv ely RNA transcript probes have been used. To reduce the effort in the prepa ration of the polynucleotides and to enhance their stability, DNA probes ma tching a part of the highly variable domain III on the 23S rRNA were constr ucted by amplification of the target region using PCR. Fluorescent labeling was achieved by incorporation of Cy3-labeled desoxyribonucleotides in the amplification. DNA polynucleotide probes were constructed for the seven val idly described Acinetobacter species. Amplified domain III rDNA of A. bauma nnii and A. calcoaceticus could be readily applied as species specific prob e. In addition, rDNA fragments could be used to recognize two groups of spe cies, one comprising A. haemolyticus, A. junii and A. radioresistens and th e other one A. lwoffii and A. johnsonii. Acinetobacter baumannii cells, som e of them occurring in filaments, could be detected by in situ hybridizatio n in native samples of activated sludge.