Acinetobacter diversity in environmental samples assessed by 16S rRNA gene PCR–DGGE fingerprinting

Abstract

A primer pair was designed to selectively amplify a fragment of the Acinetobacter 16S rRNA gene from environmental samples by PCR. 16S rRNA gene products were only obtained in PCRs with DNA from members of the genus Acinetobacter and not with DNA from other bacterial species. Denaturing gradient gel electrophoresis (DGGE) of the Acinetobacter 16S rRNA gene amplicons enabled discrimination between different Acinetobacter species. PCR using the Acinetobacter primer pair allowed detection of Acinetobacter in soil with a detection limit of 10 4 cells g −1 soil, but attachment of the GC-clamp to the forward primer resulted in a 100-fold decrease in sensitivity. Using a nested PCR approach, the detection limit could be lowered to at least 10 cells g −1 of soil. The method was applied to assess Acinetobacter diversity in soil samples originating from different historically hydrocarbon-contaminated sites. In addition, for one oil-contaminated soil, the dynamics of the Acinetobacter community in response to different treatments was monitored over time in a laboratory biostimulation experimental set-up. In all cases, bands in the DGGE fingerprints were cloned and sequenced. Environmental samples taken from a mineral oil-contaminated site and from a kerosene-contaminated site demonstrated relatively simple Acinetobacter 16S rRNA gene fingerprints with A. lwoffii and A. johnsonii as dominant members. In contrast, soils derived from MTBE- and BTEX-contaminated sites did not harbor detectable Acinetobacter populations. Although Acinetobacter was detected in the soil employed for the biostimulation experiment prior to treatment, substantial changes in its populations were observed depending on the treatment.