Comparing bacterial communities in soil is important for ecological and agricultural purposes; we investigated this process for applicability to forensic investigations, matching crime scene samples with those taken from suspects and alibi locations. We characterized the usage of molecular fingerprinting of soil bacteria in revealing biogeographic patterns. A comparison was performed of ten soil DNA extraction kits and protocols, and of the effect of soil sample storage (air dried, 4°C and -20°C). Then, we compared the effectiveness and reliability of fingerprinting methods and genes: ribosomal intergenic spacer analysis (RISA), PCR profiling of two housekeeping genes (rpoB and rpoC), real-time qPCR of six antibiotic resistance genes (tetG, tetH, blaSHV, blaTEM, ermA, sul1), and five methods using the 16S rRNA gene: phylogenetic microarrays, terminal restriction fragment length polymorphism (T-RFLP), and next-generation sequencing (NGS) with Roche 454, Illumina MiSeq and IonTorrent PGM. The highest-performing method was found to be DNA extraction with the GenomicSoil kit (Macherey Nagel) followed by RISA or T-RFLP. Sample storage had no significant effects on bacterial community. We used both RISA and T-RFLP to quantify the sensitivity of fingerprinting on samples from three soil types (rendzina, terra rossa and sand) each taken at intervals of 1, 25, 75 and 100 meters apart. Both methods, in all three soil types, displayed clear distance-decay relationships: bacterial communities were highly similar at 1m apart and increasingly different with distance. Soil samples even 25m apart could be reliably discriminated and identified. The resulting protocols are currently being tested for application in forensic scenarios.
