Evaluation of molecular techniques for identification and enumeration of Raoultella terrigena ATCC 33257 in water purifier efficacy testing

Abstract

Raoultella terrigena ATCC 33257, a representative of the coliform group, is commonly used as a challenge organism in water purifier efficacy testing. In addition to being time consuming, traditional culturing techniques and metabolic identification systems (including automated systems) also fail to accurately differentiate this organism from its closely related neighbors belonging to the Enterobacteriaceae group. Molecular-based techniques, such as real-time quantitative polymerase chain reaction (qPCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting, are preferred methods of detection because of their accuracy, reproducibility, specificity, and sensitivity, along with shorter turnaround time. ERIC-PCR performed with the 1R primer set demonstrated stable unique banding patterns (~800, ~300 bp) for R. terrigena ATCC 33257 different from patterns observed for R. planticola and R. ornithinolytica. The primer pair developed from gyraseA (gyrA) sequence of R. terrigena for the SYBR Green qPCR assay using the AlleleID(®) 7.0 primer probe design software was highly specific and sensitive for the target organism. The sensitivity of the assay was 10(1) colony forming units (CFU)/ml for whole cells and 4.7 fg with genomic DNA. The primer pair was successful in determining the concentration (5.5 ± 0.3 × 10(6) CFU/ml) of R. terrigena from water samples spiked with equal concentration of Escherichia coli and R. terrigena. Based on these results from the ERIC-PCR and the SYBR Green qPCR assay, these molecular techniques can be efficiently used for rapid identification and quantification of R. terrigena during water purifier testing.