Water environments are increasingly recognized as a conduit for the spread of antibiotic resistance, but there is need to standardize antibiotic resistance monitoring protocols to ensure ...comparability across studies. Quantitative polymerase chain reaction (qPCR) is attractive as a sensitive means of quantifying antibiotic resistance genes (ARGs) and has been applied broadly over the past two decades to various water matrices. QPCR avoids challenges and biases associated with culture-based methods, providing a reproducible and highly sensitive measure of ARGs carried across a bacterial community. However, there are numerous quality assurance and other aspects of protocols that need to be addressed to ensure that measurements are representative and comparable across studies. Here we conducted a critical review to identify gene targets that are most commonly measured by qPCR to quantify antibiotic resistance in surface water, recycled water, and wastewater and to assess corresponding protocols. Identified targets monitored in water samples included sul1, tetA, and intI1, given their abundance and tendency to correlate with anthropogenic inputs, and vanA and blaCTX-M, as more rarely detected, but highly clinically-relevant targets. We identified 117 peer-reviewed studies meeting search criteria for application of these assays to water matrices of interest and systematically assessed the corresponding protocols, including sample collection and concentration, DNA extraction, primer/probe specificity, amplification conditions, amplicon length, PCR inhibition evaluation, and limit of detection/quantification. Gene copy numbers reported across studies were further compared by assay and water matrix. Based on this comprehensive evaluation, we recommend assays, standardized workflows, and reporting for the five target genes.
Shotgun metagenomic sequencing of the collective genomic information carried across microbial communities is emerging as a powerful approach for monitoring antibiotic resistance in environmental ...matrices. Metagenomics is advantageous in that known and putative antibiotic resistance genes (ARGs) (i.e., the resistome) can be screened simultaneously without a priori selection of targets. Additionally, as new ARGs are discovered and catalogued, stored sequencing data can be reanalyzed to assess the prevalence of emerging genes or pathogens. However, best practices for metagenomic data generation and processing are needed to support comparability across space and time. To support reproducible downstream analysis, guidance is first needed with respect to sampling design, sample preservation and storage, DNA extraction, library preparation, sequencing depth, and experimental controls. Here we conducted a systematic review to assess current practices for the application of metagenomics for AR profiling of wastewater, recycled water, and surface water and to offer recommendations to support comparability in the collection, production, and analysis of resulting data. Based on integrated analysis of findings and data reported across 95 articles identified, a field to benchtop metagenomic workflow is discussed for optimizing the representativeness and comparability of generated data. Through the reanalysis of 1474 publicly-available metagenomes, appropriate sequencing depths per environment and uniform normalization strategies are provided. Further, there is opportunity to harness the quantitative capacity of metagenomics more overtly through inclusion of sequencing controls. The recommendations will amplify the overall value of the metagenomic data generated to support within and between study comparisons, now and in the future.
Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health ...information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.
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•Harmonized QA/QC procedures for SARS-CoV-2 wastewater surveillance are lacking.•Wastewater analysis protocols are not optimized for trace analysis of viruses.•False-positive and -negative errors have consequences for public health responses.•Inter-laboratory studies utilizing standardized reference materials and protocols are needed.
Vancomycin resistant enterococci (VRE) cause 20,000 infections annually in the United States, most of which are nosocomial. Recent findings of VRE in sewage-contaminated surface waters demonstrate an ...alternate route of human exposure, and a possible setting for horizontal gene exchange facilitated by plasmids and other mobile genetic elements. Maintenance of antibiotic resistance genes and proteins may, however, present a fitness cost in the absence of selective pressure, particularly in habitats such as environmental waters that are not optimal for gut-associated bacteria. Nutrient levels, which are transiently elevated following sewage spills, may also affect survival. We tested the hypotheses that nutrients and/or plasmids conferring vancomycin resistance affect Enterococcus faecium survival in river water by measuring decay of strains that differed only by their plasmid, under natural and augmented nutrient conditions. In natural river water, decay rate (log10 reduction) correlated directly with plasmid size; however, plasmid presence and size had no effect on decay rate when nutrients levels were augmented. Under natural nutrient levels, the vancomycin-resistant strain with the largest plasmid (200 kb) decayed significantly more rapidly than the plasmid-less, susceptible parent strain, in contrast to similar decay rates among strains under augmented nutrient conditions. This work is among the first to show that plasmids conferring antibiotic resistance affect fitness of Enterococcus species in secondary habitats such as surface water. The nutrient-dependent nature of the fitness cost suggests that conveyance of VRE to environmental waters in nutrient-rich sewage may prolong survival of these pathogens, providing greater opportunity for host infection and/or horizontal gene transfer.
•Vancomycin resistance plasmids exert fitness costs in surface waters.•Fitness costs are nutrient-dependent.•Size of plasmid is negatively correlated with survival.
•Metagenomic and culture methods revealed fate of ARG/ARB in water reuse systems.•Activated sludge removed the majority of ARB/ARGs compared to tertiary treatments.•Ozonation resulted in enrichment ...of multidrug ARGs.•Non-potable reuse distribution systems are vulnerable to ARG/ARB regrowth.•Resistome risk is reduced by chlorination and lowest in the recharged aquifer.
Water reuse is an essential strategy for reducing water demand from conventional sources, alleviating water stress, and promoting sustainability, but understanding the effectiveness of associated treatment processes as barriers to the spread of antibiotic resistance is an important consideration to protecting human health. We comprehensively evaluated the reduction of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in two field-operational water reuse systems with distinct treatment trains, one producing water for indirect potable reuse (ozone/biologically-active carbon/granular activated carbon) and the other for non-potable reuse (denitrification-filtration/chlorination) using metagenomic sequencing and culture. Relative abundances of total ARGs/clinically-relevant ARGs and cultured ARB were reduced by several logs during primary and secondary stages of wastewater treatment, but to a lesser extent during the tertiary water reuse treatments. In particular, ozonation tended to enrich multi-drug ARGs. The effect of chlorination was facility-dependent, increasing the relative abundance of ARGs when following biologically-active carbon filters, but generally providing a benefit in reduced bacterial numbers and ecological and human health resistome risk scores. Relative abundances of total ARGs and resistome risk scores were lowest in aquifer samples, although resistant Escherichia coli and Klebsiella pneumoniae were occasionally detected in the monitoring well 3-days downgradient from injection, but not 6-months downgradient. Resistant E. coli and Pseudomonas aeruginosa were occasionally detected in the nonpotable reuse distribution system, along with increased levels of multidrug, sulfonamide, phenicol, and aminoglycoside ARGs. This study illuminates specific vulnerabilities of water reuse systems to persistence, selection, and growth of ARGs and ARB and emphasizes the role of multiple treatment barriers, including aquifers and distribution systems.
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Disposal of fecally contaminated poultry litter by land application can deliver pathogens and fecal indicator bacteria (FIB) into receiving waters via runoff. While water quality is regulated by FIB ...enumeration, FIB testing provides inadequate information about contamination source and health risk. This microbial source tracking (MST) study compared the persistence of the Brevibacterium sp. strain LA35 16S rRNA gene (marker) for poultry litter with that of pathogens and FIB under outdoor, environmentally relevant conditions in freshwater, marine water, and sediments over 7 days. Salmonella enterica, Campylobacter jejuni, Campylobacter coli, Bacteroidales, and LA35 were enumerated by quantitative PCR (qPCR), and Enterococcus spp. and E. coli were quantified by culture and qPCR. Unlike the other bacteria, C. jejuni was not detectable after 48 h. Bacterial levels in the water column consistently declined over time and were highly correlated among species. Survival in sediments ranged from a slow decrease over time to growth, particularly in marine microcosms and for Bacteroidales. S. enterica also grew in marine sediments. Linear decay rates in water (k) ranged from -0.17 day(-1) for LA35 to -3.12 day(-1) for C. coli. LA35 levels correlated well with those of other bacteria in the water column but not in sediments. These observations suggest that, particularly in the water column, the fate of LA35 in aquatic environments is similar to that of FIB, C. coli, and Salmonella, supporting the hypothesis that the LA35 marker gene can be a useful tool for evaluating the impact of poultry litter on water quality and human health risk.
Millions of tons of fecal-contaminated poultry litter are applied to U.S. agricultural fields annually. Precipitation and irrigation facilitate transport of fecal-derived pathogens and fecal ...indicator bacteria (FIB) to groundwater. The goal of this study was to compare transport of pathogens, FIB, and a microbial source tracking marker gene for poultry litter (LA35) in a simulated soil-to-groundwater system. Nine laboratory soil columns containing four different soil types were used to evaluate microbial transport to groundwater via infiltration. Quantitative polymerase chain reaction was used to monitor Salmonella enterica Typhimurium, Escherichia coli, Enterococcus spp., Brevibacterium sp. LA35 and Bacteroidales leached from soil columns inoculated with poultry litter. S. enterica was correlated with LA35 poultry litter marker gene and FIB concentrations in column soils containing organic matter, but not in acid washed sands. In contrast, S. enterica was found to correlate with LA35 and FIB in the leachate from columns containing sand, but not with leachate from organic soil columns. The majority of recovered DNA was found in leachate of predominately sandy soil columns, and in the soil of loamy columns. At least 90% of the DNA retained in soils for each microbial target was found in the top 3cm of the column. These studies suggest that poultry litter associated pathogens and FIB are rapidly released from litter, but are influenced by complex attenuation mechanisms during infiltration, including soil type. This study advances our understanding of the potential for subsurface transport of poultry litter associated pathogens and FIB, and support the use of the LA35 marker gene for evaluating poultry litter impacts on groundwater.
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•Pathogens and FIB are released from poultry litter during rainfall and irrigation.•S. enterica in soil leachate correlates with a poultry litter marker gene and FIB.•Microorganisms decline 2 to 9 log gene copies (g soil)−1 within 12in. of soil.
The opportunistic pathogen Vibrio vulnificus occurs naturally in estuarine habitats and is readily cultured from water and oysters under warm conditions but infrequently at ambient conditions of ...<15°C. The presence of V. vulnificus in other habitats, such as sediments and aquatic vegetation, has been explored much less frequently. This study investigated the ecology of V. vulnificus in water by culture and quantitative PCR (qPCR) and in sediment, oysters, and aquatic vegetation by culture. V. vulnificus samples were taken from five sites around Tampa Bay, FL. Levels determined by qPCR and culture were significantly correlated (P = 0.0006; r = 0.352); however, V. vulnificus was detected significantly more frequently by qPCR (85% of all samples) compared to culture (43%). Culturable V. vulnificus bacteria were recovered most frequently from oyster samples (70%), followed by vegetation and sediment (∼50%) and water (43%). Water temperature, which ranged from 18.5 to 33.4°C, was positively correlated with V. vulnificus concentrations in all matrices but sediments. Salinity, which ranged from 1 to 35 ppt, was negatively correlated with V. vulnificus levels in water and sediments but not in other matrices. Significant interaction effects between matrix and temperature support the hypothesis that temperature affects V. vulnificus concentrations differently in different matrices and that sediment habitats may serve as seasonal reservoirs for V. vulnificus. V. vulnificus levels in vegetation have not been previously measured and reveal an additional habitat for this autochthonous estuarine bacterium.
We investigated the potential for a variety of environmental reservoirs to harbor or contribute fecal indicator bacteria (FIB), DNA markers of human fecal contamination, and human pathogens to a ...freshwater lake. We hypothesized that submerged aquatic vegetation (SAV), sediments, and stormwater act as reservoirs and/or provide inputs of FIB and human pathogens to this inland water. Analysis included microbial source tracking (MST) markers of sewage contamination (Enterococcus faecium esp gene, human-associated Bacteroides HF183, and human polyomaviruses), pathogens (Salmonella, Cryptosporidium, Giardia, and enteric viruses), and FIB (fecal coliforms, Escherichia coli, and enterococci). Bayesian analysis was used to assess relationships among microbial and physicochemical variables. FIB in the water were correlated with concentrations in SAV and sediment. Furthermore, the correlation of antecedent rainfall and major rain events with FIB concentrations and detection of human markers and pathogens points toward multiple reservoirs for microbial contaminants in this system. Although pathogens and human-source markers were detected in 55% and 21% of samples, respectively, markers rarely coincided with pathogen detection. Bayesian analysis revealed that low concentrations (<45 CFU × 100 ml−1) of fecal coliforms were associated with 93% probability that pathogens would not be detected; furthermore the Bayes net model showed associations between elevated temperature and rainfall with fecal coliform and enterococci concentrations, but not E. coli. These data indicate that many under-studied matrices (e.g. SAV, sediment, stormwater) are important reservoirs for FIB and potentially human pathogens and demonstrate the usefulness of Bayes net analysis for water quality assessment.
► Pathogens and fecal indicator bacteria levels were assessed in a Florida lake. ► Antecedent rainfall and stormwater negatively influenced microbial water quality. ► Bayes net modeling predicted the risk of pathogen presence. ► Fecal coliforms were better predictors of human pathogens than enterococci. ► Naturalized enterococci confounded the indicator-pathogen relationship.
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