Metabolically-active bacteria in reclaimed water and ponds revealed using bromodeoxyuridine DNA labeling coupled with 16S rRNA and shotgun sequencing

Malayil, Leena; Ramachandran, Padmini; Chattopadhyay, Suhana; Cagle, Robin; Hittle, Lauren E.; Ottesen, Andrea; Mongodin, Emmanuel F.; Sapkota, Amy R.

doi:10.1016/j.watres.2020.116185

Cited by 11 publications

(14 citation statements)

References 77 publications

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“…Extensive research on the fate and transport of ARB in water sources resulting from livestock production has been carried out ( Addison, 1984 ; Humphrey et al, 2005 ). A recent study by Malayil et al (2020) described the metabolically active bacteria diversity and abundance from reclaimed water and agricultural ponds used as alternative irrigation water sources from the mid-Atlantic United States region. The study observed that antimicrobial resistance and virulence gene profiles appeared to be more diverse and abundant in relic (inactive) DNA than in viable cells (metabolically active) in the tested water types with Actinobacteria , Flavobacterium spp., Pseudomonas spp., and Aeromonas spp.…”

Section: Resultsmentioning

confidence: 99%

“…The study observed that antimicrobial resistance and virulence gene profiles appeared to be more diverse and abundant in relic (inactive) DNA than in viable cells (metabolically active) in the tested water types with Actinobacteria , Flavobacterium spp., Pseudomonas spp., and Aeromonas spp. being the most abundant and metabolic-active microorganisms ( Malayil et al, 2020 ). Our study presented some baseline information on the prevalence of viable ARB during fresh produce production irrigated with treated municipal wastewater.…”

Section: Resultsmentioning

confidence: 99%

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Prevalence of Escherichia coli and Antibiotic-Resistant Bacteria During Fresh Produce Production (Romaine Lettuce) Using Municipal Wastewater Effluents

et al. 2021

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High demand for food and water encourages the exploration of new water reuse programs, including treated municipal wastewater usage. However, these sources could contain high contaminant levels posing risks to public health. The objective of this study was to grow and irrigate a leafy green (romaine lettuce) with treated wastewater from a municipal wastewater treatment plant to track Escherichia coli and antibiotic-resistant microorganisms through cultivation and post-harvest storage to assess their fate and prevalence. Contamination levels found in the foliage, leachate, and soil were directly (p < 0.05) related to E. coli concentrations in the irrigation water. Wastewater concentrations from 177 to 423 CFU ml−1 resulted in 15–25% retention in the foliage. Leachate and soil presented means of 231 and 116% retention, respectively. E. coli accumulation on the foliage was observed (p < 0.05) and increased by over 400% during 14-day storage (4°C). From randomly selected E. coli colonies, in all four biomass types, 81 and 34% showed resistance to ampicillin and cephalothin, respectively. Reclaimed wastewater usage for leafy greens cultivation could pose potential health risks, especially considering the bacteria found have a high probability of being antibiotic resistance. Successful reuse of wastewater in agriculture will depend on appropriate mitigation and management strategies to guarantee an inexpensive, efficient, and safe water supply.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Prevalence of Escherichia coli and Antibiotic-Resistant Bacteria During Fresh Produce Production (Romaine Lettuce) Using Municipal Wastewater Effluents

et al. 2021

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“…Three separate 500 mL aliquots of each water sample were subjected to BrdU treatment using our previously published method [ 44 ]. Briefly, 100 μL of 100 mM BrdU was added per 500 mL of water and incubated for 24 h in the dark at room temperature along with the non-BrdU labeled samples.…”

Section: Methodsmentioning

confidence: 99%

“…Immunocapture and isolation of BrdU-labeled were performed using our previously published methods [ 44 ]. Briefly, sheared and denatured herring sperm DNA (HS DNA) and monoclonal anti-BrdU (α-BrdU) antibody was mixed in a 1:9 ratio and incubated for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…BrdU is a synthetic thymidine analog that incorporates into replicating DNA; therefore, bacteria detected in BrdU-treated samples are interpreted to be metabolically-active, viable members of the tested bacterial community [ 77 ]. This BrdU labeling technique has been used to identify the metabolically-active fraction of bacteria present in aquatic and soil environments [ 44 , 75 , 77 ]. Thus, the goal of this study was to evaluate whether coupling 5-bromo-2′-deoxyuridine- (BrdU) labeling with next-generation sequencing methods could enable the detection of VBNC vibrios , as well as the differentiation between metabolically-active and dead organisms in nontraditional irrigation water sources.…”

Section: Introductionmentioning

confidence: 99%

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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

Malayil

Chattopadhyay

Mongodin

et al. 2021

Environmental Microbiome

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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.

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SHF confers radioresistance in colorectal cancer by the regulation of mitochondrial DNA copy number

Zhu

Gong

et al. 2022

Clin Exp Med

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Metabolically-active bacteria in reclaimed water and ponds revealed using bromodeoxyuridine DNA labeling coupled with 16S rRNA and shotgun sequencing

Cited by 11 publications

References 77 publications

Prevalence of Escherichia coli and Antibiotic-Resistant Bacteria During Fresh Produce Production (Romaine Lettuce) Using Municipal Wastewater Effluents

Prevalence of Escherichia coli and Antibiotic-Resistant Bacteria During Fresh Produce Production (Romaine Lettuce) Using Municipal Wastewater Effluents

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

SHF confers radioresistance in colorectal cancer by the regulation of mitochondrial DNA copy number

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