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Abstract Nontraditional irrigation water sources (e.g., recycled water, brackish water) may harbor human pathogens, includingVibriospp., 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-activeVibriospp.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-activeVibriospp. at all sampling sites. Whereas, the culture-based method only detected vibrios at three of the four sites. We observed the presence ofV. cholerae,V. vulnificus, andV. parahaemolyticususing both methods, whileV. aesturianusandV. shiloniiwere detected only through our labeling/sequencing approach. Multiple other pathogens of concern to human health were also identified through our labeling/sequencing approach includingP. shigelloides,B. cereusandE. cloacae. Most importantly, 16S rRNA sequencing of BrdU-labeled samples resulted inVibriospp. detection even when our culture-based methods resulted in negative detection. This suggests that our novel approach can effectively detect metabolically-activeVibriospp. that may have been present in a VBNC state, refining our understanding of the prevalence of vibrios in nontraditional irrigation waters.
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This content will become publicly available on May 1, 2026
Comparison of Two DNA Labeling Dyes Commonly Used to Detect Metabolically Active Bacteria
Bacteria are ubiquitous in the environment and critical to human health and disease, yet only a small fraction can be identified through standard culture methods. Advances in next-generation sequencing techniques have improved bacterial identification, but these DNA-based methods cannot distinguish live bacteria from relic DNA. Recently, DNA-labeling dyes (e.g., 5-bromo-2′-deoxyuridine [BrdU] and propidium monoazide [PMA]) have been used to detect metabolically active bacteria in different sample types. Here, we compare BrdU and PMA in combination with 16SrRNA gene sequencing to characterize metabolically active bacteria in two different sample types: (1) manufactured products (n = 78; cigarettes, hookah, and little cigar) and (2) natural samples (n = 186; rainwater, soil, and produce). Metabolically active bacterial communities identified in BrdU-labeled samples had lower alpha diversity than that of PMA-treated and non-treated samples. Pseudomonas, Sphingomonas, Enterobacter, and Acinetobacter were observed in all the samples tested. Irrespective of sample type, Pseudomonas was predominant in BrdU-treated samples, while Acinetobacter was more abundant in non-treated samples compared to PMA-treated samples. We also observed that PMA-treated samples tend to overestimate the metabolically active bacterial fraction compared to BrdU-treated samples. Overall, our study highlights how different labeling techniques influence bacterial community analysis findings, underscoring the need for careful selection of labeling approaches when assessing environmental samples.
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- Award ID(s):
- 1828910
- PAR ID:
- 10601107
- Publisher / Repository:
- Microorganisms
- Date Published:
- Journal Name:
- Microorganisms
- Volume:
- 13
- Issue:
- 5
- ISSN:
- 2076-2607
- Page Range / eLocation ID:
- 1015
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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