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Nontraditional irrigation water sources (e.g., recycled water, brackish water) may harbor human pathogens, including  Vibrio  spp., that could be present in a viable-but-nonculturable (VBNC) state, stymieing current culture-based detection methods. To overcome this challenge, we coupled 5-bromo-2′-deoxyuridine (BrdU) labeling, enrichment techniques, and 16S rRNA sequencing to identify metabolically-active  Vibrio  spp .  in nontraditional irrigation water (recycled water, pond water, non-tidal freshwater, and tidal brackish water). Our coupled BrdU-labeling and sequencing approach revealed the presence of metabolically-active  Vibrio  spp. at all sampling sites. Whereas, the culture-based method only detected vibrios at three of the four sites. We observed the presence of  V. cholerae ,  V. vulnificus , and  V. parahaemolyticus  using both methods, while  V. aesturianus  and  V. shilonii  were detected only through our labeling/sequencing approach. Multiple other pathogens of concern to human health were also identified through our labeling/sequencing approach including  P. shigelloides ,  B. cereus  and  E. cloacae . Most importantly, 16S rRNA sequencing of BrdU-labeled samples resulted in  Vibrio  spp. detection even when our culture-based methods resulted in negative detection. This suggests that our novel approach can effectively detect metabolically-active  Vibrio  spp. that may have been present in a VBNC state, refining our understanding of the prevalence of vibrios in nontraditional irrigation waters.   Supplementary Information  The online version contains supplementary material available at 10.1186/s40793-021-00382-1.

environmental microbiome

Coupled DNA-labeling and sequencing approach enables the detection of viable-but-non-culturable Vibrio spp. in irrigation water sources in the Chesapeake Bay watershed