Development of two quantitative PCR assays for the detection of emerging opportunistic human pathogens belonging to the genus Phytobacter in routine diagnostics

The genus Phytobacter (fam. Enterobacteriaceae) includes species like Phytobacter diazotrophicus and Phytobacter ursingii, which have emerged as opportunistic human pathogens, particularly affecting vulnerable populations such as pre-term infants and immunocompromised patients. Traditional biochemical and molecular methods have struggled to accurately identify Phytobacter species in clinical diagnostics. This study addresses the issue by developing and validating two quantitative PCR (qPCR) assays using SYBR® Green I and TaqMan® technologies, targeting the nitrogen fixation regulatory gene (nifL) of Phytobacter spp. The SYBR® Green I assay showed a detection limit of a single cell per reaction, while the TaqMan® assay was easier to interpret due to the absence of background noise. These assays, validated with clinical isolates from Brazil, identified multiple new Phytobacter isolates, including a potentially novel species, providing improved diagnostic tools for detecting Phytobacter spp. and aiding in better clinical management.