Search

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. [ABSTRACT FROM AUTHOR]

Awareness of the need for surveillance of antimicrobial resistance (AMR) in water environments is growing, but there is uncertainty regarding appropriate monitoring targets. Adapting culture-based fecal indicator monitoring to include antibiotics in the media provides a potentially low-tech and accessible option, while quantitative polymerase chain reaction (qPCR) targeting key genes of interest provides a broad, quantitative measure across the microbial community. The purpose of this study was to compare findings obtained from the culture of cefotaxime-resistant (cefR) Escherichia coli with two qPCR methods for quantification of antibiotic resistance genes across wastewater, recycled water, and surface waters. The culture method was a modification of US EPA Method 1603 for E. coli, in which cefotaxime is included in the medium to capture cefR strains, while qPCR methods quantified sul1 and intI1. A common standard operating procedure for each target was applied to samples collected by six water utilities across the United States and processed by two laboratories. The methods performed consistently, and all three measures reflected the same overarching trends across water types. The qPCR detection of sul1 yielded the widest dynamic range of measurement as an AMR indicator (7-log versus 3.5-log for cefR E. coli), while intI1 was the most frequently detected target (99% versus 96.5% and 50.8% for sul1 and cefR E. coli, respectively). All methods produced comparable measurements between labs (p < 0.05, Kruskal–Wallis). Further study is needed to consider how relevant each measure is to capturing hot spots for the evolution and dissemination of AMR in the environment and as indicators of AMR-associated human health risk. [ABSTRACT FROM AUTHOR]

Fecal indicator bacteria (FIB: Escherichia coli and enterococci) are used to assess recreational water quality. Viral indicators (i.e., somatic and F+ coliphage), could improve the prediction of viral pathogens in recreational waters, however, the impact of environmental factors, including the effect of predatory protozoa source, on their survival in water is poorly understood. We investigated the effect of lakewater or wastewater protozoa, on the decay (decreasing concentrations over time) of culturable FIB and coliphages under sunlight and shaded conditions. FIB decay was generally greater than the coliphages and was more rapid when indicators were exposed to lake vs. wastewater protozoa. F+ coliphage decay was the least affected by experimental variables. Somatic coliphage decayed fastest in the presence of wastewater protozoa and sunlight, though their decay under shaded conditions was-10-fold less than F+ after 14 days. The protozoa source consistently contributed significantly to the decay of FIB, and somatic, though not the F+ coliphage. Sunlight generally accelerated decay, and shade reduced somatic coliphage decay to the lowest level among all the indicators. Differential responses of FIB, somatic, and F+ coliphages to environmental factors support the need for studies that address the relationship between the decay of coliphages and viral pathogens under environmentally relevant conditions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aims: Beach water quality is regulated by faecal indicator bacteria levels, sand is not, despite known human health risk from exposure to beach sand. We compared the performance of three methods to extract bacterial DNA from beach sand as a step toward a standard method. Methods and results: The analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus direct extraction using PowerSoil™ or PowerMax Soil™ kits. The slurry method had the lowest limit of detection at 20–80 gene copies g−1, recovered significantly more DNA, and the only method that detected Enterococcus by qPCR in all samples; therefore, the only method used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the microbial source tracking marker HF183 from wastewater spiked marine and freshwater beach sand was 100.8% and 64.1%, respectively, but varied, indicating that the mixing protocol needs improvement. Conclusions: Among the three methods, the slurry method had the best analytical sensitivity and produced extracts that were useful for culture or molecular analysis. Significance and impact of study: Standardization of methods for extraction of bacterial DNA from sand facilitates comparisons among studies, and ultimately contributes to the safety of recreational beaches. [ABSTRACT FROM AUTHOR]

A common operational problem in leachate collection systems is clogging due to the formation of deposits within pore spaces and collection pipes. The role of co-disposal of municipal solid waste [...]

The diversity and stability of Escherichia coli populations within the feces of individuals belonging to three mammalian host groups (humans, horses, and beef cattle) are investigated. The high genetic diversity of Escherichia coli populations, variations in diversity among hosts, and temporal variability all indicate complex population dynamics that impact the usefulness of Escherichia coli as a water indicator and its use in microbial source tracking studies.

PCR is used to screen DNA polymorphisms in strains from environmental or clinical sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P

The validity of using indicator organisms was tested to predict the presence of absence of pathogens at six wastewater reclamation facilities. A major goal of wastewater reclamation facilities is to reduce pathogen loads in order to decrease public health risks associated with exposure.

Indicator organisms that reside in the gastrointestinal tracts of humans and animals are used worldwide to monitor water quality. The results have implications for the accuracy of microbial source tracking methods, modeling of microbial populations in water, and efficacy of regulatory standards for protection of water quality.

Escherichia coli is a promising subject for globally coordinated surveillance of antimicrobial resistance (AMR) in water environments due to its clinical relevance and widespread use as an indicator of fecal contamination. Cefotaxime-resistant E. coli was recently evaluated favorably for this purpose by the World Health Organization TriCycle Protocol, which specifies tryptone bile x-glucuronide (TBX) medium and incubation at 35°C. We assessed comparability with the U.S. Environmental Protection Agency-approved method for E. coli quantification, which uses membrane-thermotolerant E. coli (mTEC) agar and incubation at 44.5°C, in terms of recovery of E. coli and cefotaxime-resistant E. coli from wastewater influent and surface waters. Total E. coli concentrations in wastewater influent were 106-108 CFU/100 mL, while cefotaxime-resistant E. coli were ~100-fold lower. Total E. coli in surface waters were ~10² CFU/100 mL, and cefotaxime-resistant isolates were near the limit of detection (0.4 CFU/100 mL). Total and putative cefotaxime-resistant E. coli concentrations did not differ significant ly between media or by incubation method; however, colonies isolated on mTEC were more frequently confirmed to species (97.1%) compared to those from TBX (92.5%). Incubation in a water bath at 44.5°C significantly decreased non-specific background growth and improved confirmation frequency on both media (97.4%) compared to incubation at 35°C (92.3%). This study helps to advance globally coordinated AMR in water environments and suggests that the TriCycle Protocol is adaptable to other standard methods that may be required in different locales, while also offering a means to improve specificity by decreasing the frequency of false-positive identification of cefotaxime-resistant E. coli by modifying incubation conditions. [ABSTRACT FROM AUTHOR]

Combination of hollow-fiber ultrafiltration (HFUF) and PCR are utilized to detect human-associated microbial source tracking marker genes in large volumes of fresh and estuarine Florida water. HFUF which facilitated the marker detection demonstrated the ability of HFUF to allow detection of diluted targets by PCR in a variety of water types.

The presence of all three classes of pathogens (viral, protozoan and bacterial) as well as indicator microbes at a non-point source recreational beach is assessed. The analysis of the various microbes under different targeted environmental conditions has included the evaluation of the sources of microbial contaminants and pathogens and the effectiveness of various analytical methods for microbe detection.

A TaqMan quantitative PCR (QPCR) assay is developed and optimized based on the conserved T antigen in order to quantify and detect two human polyomaviruses (HPyVs), JC virus and BK virus. The use of this quantitative assay in water quality research might help regulatory agencies in identifying sources of water pollution for improved remediation of contaminated waters and thus protecting humans from exposure to pathogens.

Real-time PCR assays developed for detecting and differentiating two 16S rRNA variants of Vibrio vulnificus in oysters and water is reported. These assays were found to be specific and sensitive in differentiating the strains of V. vulnificus in oysters without requiring its isolation and may be useful in quick detection of pathogens in shellfish and water.

The development, performance and validation of D source tracking (MST) methods are described.

A method developed to concentrate and extract human polyomaviruses (HPyVs) DNA, which are used as indicators of human fecal pollution, from environmental water samples and to amplify it by nested PCR is presented. The sensitivity, specificity and correlation with other human-associated markers have indicated that the HPyV assay can be a useful predictor of human fecal pollution in environmental waters and an important component of the microbial-source-tracking toolbox.