Viability-based quantification of antibiotic resistance genes and human fecal markers in wastewater effluent and receiving waters

Eramo, Alessia; Medina, William R. Morales; Fahrenfeld, Nicole

doi:10.1016/j.scitotenv.2018.11.325

Cited by 27 publications

(15 citation statements)

References 36 publications

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“…sul 1 was the most commonly detected ARG transcript in the sewer samples. This has consistently been reported as the most abundant ARG among all matrices in sewer system (wastewater [ 3 – 5 , 7 , 32 ], sewer sediment [ 2 , 5 ], and biofilms [ 2 , 7 ]) including in the paired DNA samples for this study ranging from − 1 to − 2 Log10 copies per copy of 16S rRNA gene [ 5 ]. tet (G) and bla TEM have also been commonly detected in sewer bacterial communities [ 2 – 5 , 7 , 32 , 33 ].…”

Section: Discussionsupporting

confidence: 73%

“…A previous study on soil samples from alpine forests, alpine tundra, grasslands, and wetlands showed that in some soil ecosystems, extracellular DNA and DNA from dead cells can inflate the prokaryotic community richness and misestimate the relative abundances of bacterial taxa [ 47 ]. Another study in chlorinated wastewater effluent showed an average ratio of ~ 0.3 of 16S rRNA genes from viable cells to 16S rRNA genes from dead cells and extracellular DNA [ 3 ]; this is expected to be higher in untreated wastewater influent. This could explain why more sequences of some of the relevant taxa containing human pathogens were detected in the rRNA gene microbiome than in the rRNA transcript microbiomes (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…qPCR targeting selected ARGs ( sul 1[ 18 ], tet (G)[ 19 ], tet (W)[ 20 ], tet (O)[ 20 ], erm F[ 21 ], NDM-1[ 22 ], van A[ 23 ], and bla TEM [ 24 ]) and traditional PCR targeting atp E [ 25 ], a functional gene present in non-tuberculous Mycobacterium spp., was performed on the cDNA that was synthesized from the mRNA of each sample. These ARGs encoding for sulfonamide, macrolide, tetracycline, beta-lactam, and vancomycin antibiotic resistance were selected due to their common abundance in sewer system matrices [ 3 – 5 ]. NDM-1 was selected because some bacterial species with this functional gene have been recently classified as an “urgent threat” by the US CDC [ 1 ].…”

Section: Methodsmentioning

confidence: 99%

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Metabolically Active Prokaryotes and Actively Transcribed Antibiotic Resistance Genes in Sewer Systems: Implications for Public Health and Microbially Induced Corrosion

2021

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Sewer systems are reservoirs of pathogens and bacteria carrying antibiotic resistance genes (ARGs). However, most recent high-throughput studies rely on DNA-based techniques that cannot provide information on the physiological state of the cells nor expression of ARGs. In this study, wastewater and sewer sediment samples were collected from combined and separate sanitary sewer systems. The metabolically active prokaryote community was evaluated using 16S rRNA amplicon sequencing and actively transcribed ARG abundance was measured using mRNA RT-qPCR. Three ( sul 1, bla TEM , tet (G)) of the eight tested ARGs were quantifiable in select samples. Sewer sediment samples had greater abundance of actively transcribed ARGs compared to wastewater. Microbiome analysis showed the presence of metabolically active family taxa that contain clinically relevant pathogens (Pseudomonadaceae, Enterobacteraceae, Streptococcaceae, Arcobacteraceae, and Clostridiaceae) and corrosion-causing prokaryotes (Desulfobulbaceae and Desulfovibrionaceae) in both matrices. Spirochaetaceae and methanogens were more common in the sediment matrix while Mycobacteraceae were more common in wastewater. The microbiome obtained from 16S rRNA sequencing had a significantly different structure from the 16S rRNA gene microbiome. Overall, this study demonstrates active transcription of ARGs in sewer systems and provides insight into the abundance and physiological state of taxa of interest in the different sewer matrices and sewer types relevant for wastewater-based epidemiology, corrosion, and understanding the hazard posed by different matrices during sewer overflows. Supplementary Information The online version contains supplementary material available at 10.1007/s00248-021-01775-y.

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Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Metabolically Active Prokaryotes and Actively Transcribed Antibiotic Resistance Genes in Sewer Systems: Implications for Public Health and Microbially Induced Corrosion

2021

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“…For ARG quantification, amplificationdependent methods such as quantitative PCR (qPCR), high-throughput qPCR (HT-qPCR) and digital PCR (dPCR) are particularly useful, in part due to their ease of execution, robustness, specificity and sensitivity. qPCR can provide information on the abundance of the targeted ARGs in different genetic contexts including viable bacteria, mobile DNA fragments like MGEs and "free" environmental DNA (extracellular DNA), depending on the DNA extraction technique(Dong et al, 2019;Eramo et al, 2019). For example, propidium monoazide can be used to remove DNA from dead cells and extracellular DNA in the sample during the extraction process, thus allowing for the obtainment of DNA from live cells only(Wagner et al, 2008).Similarly, it may also be possible to selectively target plasmids and separate them from chromosomal DNA.…”

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confidence: 99%

Monitoring antibiotic resistance genes in wastewater treatment: Current strategies and future challenges

Nguyen

et al. 2021

Science of The Total Environment

176

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Antimicrobial resistance (AMR) is a growing threat to human and animal health.Progress in molecular biology has revealed new and significant challenges for AMR mitigation given the immense diversity of antibiotic resistance genes (ARGs), the complexity of ARG transfer, and the broad range of omnipresent factors contributing to AMR. Municipal, hospital and abattoir wastewater are collected and treated in wastewater treatment plants (WWTPs), where the presence of diverse selection pressures together with a highly concentrated consortium of pathogenic/commensal microbes create favourable conditions for the transfer of ARGs and proliferation of antibiotic resistant bacteria (ARBs). The recent emergence ARBs and ARGs as well as their potential health effects have re-defined the role of WWTPs as a focal point in the fight against AMR. By reviewing the occurrence of ARGs in wastewater and sludge and the current technologies used to quantify ARGs and identify antibiotic resistant bacteria (ARB), this paper provides a research roadmap to address existing challenges in AMR control via wastewater treatment. Wastewater treatment is a double-edged sword that can act as either a pathway for AMR spread or as a barrier to reduce the environmental release of anthropogenic AMR. State of the art ARB identification technologies, such as metagenomic sequencing and fluorescence-activated cell sorting, have enriched ARG/ARB databases, unveiled keystone species in AMR networks, and improved the resolution of AMR dissemination models. Data and information provided in this review highlight significant knowledge gaps. These include inconsistencies in ARG reporting units, lack of ARG/ARB monitoring surrogates, lack of a standardised protocol for determining ARG removal via wastewater treatments, and the inability to support appropriate risk assessment. This is due to a lack of standard monitoring targets and agreed threshold values, and paucity of information on the ARG-pathogen host relationship and potential risk evolution. These research gaps need to be addressed and research 3 findings need to be transformed into practical guidance for WWTP operators to enable effective progress towards mitigating the evolution and spread of AMR.

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“…When used in conjunction with a DNA-binding dye, such as propidium monoazide (PMA), extracellular and non-viable cell DNA are covalently cross-linked by the dye and hindered from detection in the downstream PCR and sequencing processes, allowing the detection of the viable microbes in specific (Kibbee and Örmeci, 2017 ). With more studies unraveling the possible interferences and optimizing the procedures of PMA treatment, the use of PMA in complex environmental sample matrices, such as sewage, has become more common in the past decade (Li et al, 2014b ; Eramo et al, 2019 ). Applications of PMA-coupled qPCR or sequencing have discovered a great variety of viable cells in disinfected effluents (Pang et al, 2016 ; Kibbee and Örmeci, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

Strategy to Evaluate Changes in Bacterial Community Profiles and Bacterial Pathogen Load Reduction After Sewage Disinfection

Tang

Lau

2022

Front. Microbiol.

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Sewage effluent discharge is a major source of pathogenic contamination to the environment. The disinfection process is critical for the elimination of pathogens in sewage. In this study, we examined the impact of chlorine disinfection on the total, viable, and culturable populations of indicator bacteria, pathogens, and bacterial communities in two contrasting types of effluents (primarily treated saline and secondarily treated freshwater). Effluents collected bimonthly over 1 year were examined using cultivation, quantitative PCR (qPCR), and 16S rRNA gene amplicon sequencing coupled with or without propidium monoazide (PMA) treatment. The results showed that each type of effluent was characterized by a specific set of representative genera before disinfection. Salinity appeared to be the major abiotic factor associated with the differences in bacterial community compositions. The pathogen analysis pipeline revealed over 20 viable clinically important pathogenic species in the effluents. Although the bacterial communities differed markedly between the two types of effluents before disinfection, the species of pathogens persisting after disinfection were similar, many of them were members of Enterobacter and Vibrio. The relative abundances of all pathogens identified in the amplicon sequences were multiplied by the 16S rRNA gene copy numbers of total bacteria detected by PMA-qPCR to estimate their concentrations. Pathogens remained viable after disinfection reached 8 log10 16S rRNA copies ml−1 effluent. Meanwhile, around 80 % of the populations of three indicator bacteria including Escherichia coli, Enterococcus, and Bacteroidales were viable after disinfection, but over 99 % of the viable E. coli and Enterococcus were in the non-culturable state. We estimated the total pathogen load by adding the concentrations of all viable pathogens and examined their correlations with indicator bacteria of different types, physiological states, and effluents. The results showed that the PMA-qPCR measurement of E. coli is a reliable proxy of bacterial pathogen loads in both types of effluents. The utility of viable indicator bacteria as a biological index to assess the overall bacteriological hazards in effluents is discussed.

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Viability-based quantification of antibiotic resistance genes and human fecal markers in wastewater effluent and receiving waters

Cited by 27 publications

References 36 publications

Metabolically Active Prokaryotes and Actively Transcribed Antibiotic Resistance Genes in Sewer Systems: Implications for Public Health and Microbially Induced Corrosion

Metabolically Active Prokaryotes and Actively Transcribed Antibiotic Resistance Genes in Sewer Systems: Implications for Public Health and Microbially Induced Corrosion

Monitoring antibiotic resistance genes in wastewater treatment: Current strategies and future challenges

Strategy to Evaluate Changes in Bacterial Community Profiles and Bacterial Pathogen Load Reduction After Sewage Disinfection

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