Simultaneous enumeration of different bacteria using reverse sample genome probing technique

Quantitative reverse sample genome probing (RSGP) with λDNA as an internal standard was used to enumerate the total numbers of Rhizobium sp. CCRC 13560, Rhizobium meliloti CCRC 13516 and Bradyrhizobium sp. CCRC 13585. K λ / Kx ratios varied between the three species but also in response to the amounts of λDNA or genomic DNA used in the labeling mixture or fixed upon the membrane. Comparative enumerations of pure cultures revealed higher counts using genomic probing as compared with growth-based colony forming units (CFU; 3.4±1.7-fold higher for R. meliloti, 6.4±7.8-fold higher for Rhizobium sp. and 0.34±0.17-fold higher for Bradyrhizobium sp.). In mixed cultures, the estimated cell numbers using genomic probing were 126±172-, 85±83- and 4.0±3.4-fold higher (same respective order) than the growth-based assay. By replacing the k λ / k x ratio with k′ λ / k′ x (slope from signal intensity of differently diluted λDNA/slope from signal intensity of differently diluted target DNA× f x / f λ ), significant improvement in the accuracy of the estimation was achieved. The calculated cell numbers via the genomic probe technique were 0.99±0.13-, 1.25±0.23- and 0.18±0.11-fold higher than the respective CFUs in pure cultures of R. meliloti, Rhizobium sp. and Bradyrhizobium sp. In samples containing mixed populations, the estimated numbers from genomic probing were 1.25±0.51-, 45.9±14.8- and 0.27±0.07-fold higher than the CFU-derived cell count (same respective order).