Evaluating the impact of sample storage, handling, and technical ability on the decay and recovery of SARS-CoV-2 in wastewater

Wastewater based epidemiology (WBE) is useful for tracking and monitoring the level of disease prevalence in a community and has been used extensively to complement clinical testing during the current COVID-19 pandemic. Despite the numerous benefits, sources of variability in sample storage, handling, and processing methods can make WBE data difficult to generalize. We performed an experiment to determine sources of variability in WBE data including the impact of storage time, handling, and processing techniques on the concentration of SARS-CoV-2 in wastewater influent from three wastewater treatment plants (WWTP) in North Carolina over 19 days. The SARS-CoV-2 concentration in influent samples held at 4°C did not degrade significantly over the 19-day experiment. Heat pasteurization did not significantly impact the concentration of SARS-CoV-2 at two of the three WWTP but did reduce viral recovery at the WWTP with the smallest population size served. On each processing date, one filter from each sample was processed immediately while a replicate filter was frozen at -80°C. Once processed, filters previously frozen were found to contain slightly higher concentrations (<0.2 log copies/L) than their immediately processed counterparts, indicating freezing filters is a viable method for delayed quantification and may even improve recovery at WWTP with low viral concentrations. Investigation of factors contributing to variability during sample processing indicated that analyst experience level contributed significantly (p<0.001) to accepted droplet generation while extraction efficiency and reverse transcription efficiency contributed significantly (p<0.05) to day-to-day SARS-CoV-2 variability. This study provides valuable practical information for minimizing decay and/or loss of SARS CoV-2 in wastewater influent while adhering to safety procedures, promoting efficient laboratory workflows, and accounting for sources of variability

Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

ABSTRACT The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium , although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water

Halobacteriovorax, an underestimated predator on bacteria: potential impact relative to viruses on bacterial mortality

Predation on bacteria and accompanying mortality are important mechanisms in controlling bacterial populations and recycling of nutrients through the microbial loop. The agents most investigated and seen as responsible for bacterial mortality are viruses and protists. However, a body of evidence suggests that predatory bacteria such as the Halobacteriovorax (formerly Bacteriovorax), a Bdellovibrio-like organism, contribute substantially to bacterial death. Until now, conclusive evidence has been lacking. The goal of this study was to better understand the contributors to bacterial mortality by addressing the poorly understood role of Halobacteriovorax and how their role compares with that of viruses. The results revealed that when a concentrated suspension of Vibrio parahaemolyticus was added into microcosms of estuarine waters, the native Halobacteriovorax were the predators that responded first and most rapidly. Their numbers increased by four orders of magnitude, whereas V. parahaemolyticus prey numbers decreased by three orders of magnitude. In contrast, the extant virus population showed little increase and produced little change in the prey density. An independent experiment with stable isotope probing confirmed that Halobacteriovorax were the predators primarily responsible for the mortality of the V. parahaemolyticus. The results show that Halobacteriovorax have the potential to be significant contributors to bacterial mortality, and in such cases, predation by Halobacteriovorax may be an important mechanism of nutrient recycling. These conclusions add another dimension to bacterial mortality and the recycling of nutrients

Using detrending to assess SARS-CoV-2 wastewater loads as a leading indicator of fluctuations in COVID-19 cases at fine temporal scales: Correlations across twenty sewersheds in North Carolina

Wastewater surveillance emerged during the COVID-19 pandemic as a novel strategy for tracking the burden of illness in communities. Previous work has shown that trends in wastewater SARS-CoV-2 viral loads correlate well with reported COVID-19 case trends over longer time periods (i.e., months). We used detrending time series to reveal shorter sub-trend patterns (i.e., weeks) to identify leads or lags in the temporal alignment of the wastewater/case relationship. Daily incident COVID-19 cases and twice-weekly wastewater SARS-CoV-2 viral loads measured at 20 North Carolina sewersheds in 2021 were detrended using smoothing ranges of ∞, 16, 8, 4 and 2 weeks, to produce detrended cases and wastewater viral loads at progressively finer time scales. For each sewershed and smoothing range, we calculated the Spearman correlation between the cases and the wastewater viral loads with offsets of -7 to +7 days. We identified a conclusive lead/lag relationship at 15 of 20 sewersheds, with detrended wastewater loads temporally leading detrended COVID-19 cases at 11 of these sites. For the 11 leading sites, the correlation between wastewater loads and cases was greatest for wastewater loads sampled at a median lead time of 6 days before the cases were reported. Distinct lead/lag relationships were the most pronounced after detrending with smoothing ranges of 4–8 weeks, suggesting that SARS-CoV-2 wastewater viral loads can track fluctuations in COVID-19 case incidence rates at fine time scales and may serve as a leading indicator in many settings. These results could help public health officials identify, and deploy timely responses in, areas where cases are increasing faster than the overall pandemic trend

Decadal monitoring reveals an increase in Vibrio spp. concentrations in the Neuse River Estuary, North Carolina, USA.

A decade long study was conducted to investigate the ecological, biological, and temporal conditions that affect concentrations of Vibrio spp. bacteria in a well-studied lagoonal estuary. Water samples collected from the Neuse River Estuary in eastern North Carolina from 2004-2014 (with additional follow-up samples from Fall of 2018) were analyzed to determine Vibrio spp. concentrations, as well as the concentrations of inorganic and organic nutrients, fecal indicator bacteria, phytoplankton biomass, and a wide range of other physio-chemical estuarine parameters. A significant increase in Vibrio spp. was observed to occur in the estuary over the examined period. Strikingly, over this long duration study period, this statistically significant increase in total culturable Vibrio spp. concentrations does not appear to be correlated with changes in salinity, temperature, or dissolved oxygen, the three most commonly cited influential factors that predict estuarine Vibrio spp. abundance. Furthermore, shorter term (~3 years) data on specific Vibrio species (V. vulnificus and V. parahaemolyticus)show that while Vibrio spp. are increasing overall as a genus, the numbers of some key potentially pathogenic species are decreasing as a part of the total population, further supporting the concept that quantification of the entire genus is not a worthwhile use of resources toward predicting levels of specific potentially pathogenic species of public health concern. The significant increase in this concentration of Vibrio spp. in the studied estuary appears to be related to nitrogen and carbon in the system, indicating a continued need for further research

Lessons Learned from Implementing a Wet Laboratory Molecular Training Workshop for Beach Water Quality Monitoring

UA Open Access Publishing FundRapid molecular testing methods are poised to replace many of the conventional, culturebased tests currently used in fields such as water quality and food science. Rapid qPCR methods have the benefit of being faster than conventional methods and provide a means to more accurately protect public health. However, many scientists and technicians in water and food quality microbiology laboratories have limited experience using these molecular tests. To ensure that practitioners can use and implement qPCR techniques successfully, we developed a week long workshop to provide hands-on training and exposure to rapid molecular methods for water quality management. This workshop trained academic professors, government employees, private industry representatives, and graduate students in rapid qPCR methods for monitoring recreational water quality. Attendees were immersed in these new methods with hands-on laboratory sessions, lectures, and one-on-one training. Upon completion, the attendees gained sufficient knowledge and practice to teach and share these new molecular techniques with colleagues at their respective laboratories. Key findings from this workshop demonstrated: 1) participants with no prior experience could be effectively trained to conduct highly repeatable qPCR analysis in one week; 2) participants with different desirable outcomes required exposure to a range of different platforms and sample processing approaches; and 3) the collaborative interaction amongst newly trained practitioners, workshop leaders, and members of the water quality community helped foster a cohesive cohort of individuals which can advocate powerful cohort for proper implementation of molecular methods.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Lessons learned from implementing a wet laboratory molecular training workshop for beach water quality monitoring.

Rapid molecular testing methods are poised to replace many of the conventional, culture-based tests currently used in fields such as water quality and food science. Rapid qPCR methods have the benefit of being faster than conventional methods and provide a means to more accurately protect public health. However, many scientists and technicians in water and food quality microbiology laboratories have limited experience using these molecular tests. To ensure that practitioners can use and implement qPCR techniques successfully, we developed a week long workshop to provide hands-on training and exposure to rapid molecular methods for water quality management. This workshop trained academic professors, government employees, private industry representatives, and graduate students in rapid qPCR methods for monitoring recreational water quality. Attendees were immersed in these new methods with hands-on laboratory sessions, lectures, and one-on-one training. Upon completion, the attendees gained sufficient knowledge and practice to teach and share these new molecular techniques with colleagues at their respective laboratories. Key findings from this workshop demonstrated: 1) participants with no prior experience could be effectively trained to conduct highly repeatable qPCR analysis in one week; 2) participants with different desirable outcomes required exposure to a range of different platforms and sample processing approaches; and 3) the collaborative interaction amongst newly trained practitioners, workshop leaders, and members of the water quality community helped foster a cohesive cohort of individuals which can advocate powerful cohort for proper implementation of molecular methods

Comparison of Rapid Quantitative PCR-Based and Conventional Culture-Based Methods for Enumeration of Enterococcus spp. and Escherichia coli in Recreational Waters▿

Recreational water quality is currently monitored using culture-based methods that require 18 to 96 h for results. Quantitative PCR (QPCR) methods that can be completed in less than 2 h have been developed, but they could yield different results than the conventional methods. We present two studies in which samples were processed simultaneously for Enterococcus spp. and Escherichia coli using two culture-based methods (EPA method 1600 and Enterolert/Colilert-18) and QPCR. The proprietary QPCR assays targeted the 23S rRNA (Enterococcus spp.) and uidA (E. coli) genes and were conducted using lyophilized beads containing all reagents. In the first study, the QPCR method developers processed 54 blind samples that were inoculated with sewage or pure cultures or were ambient beach samples. The second study involved 163 samples processed by water quality personnel. The correlation between results of QPCR and EPA 1600 during the first study (r2) was 0.69 for Enterococcus spp., which was less than that observed between the culture-based methods (r2, 0.87). During the second study, the correlations were similar. No false positives occurred in either study when QPCR-based assays were used with blank samples. Levels of reproducibility measured through coefficients of variation were similar for results by Enterococcus QPCR and culture-based methods during both studies but were higher for E. coli QPCR results in the first study. Regarding the concentration at which beach management decisions are issued in the State of California, the agreement between results of Enterococcus QPCR and EPA method 1600 was 88%, compared to 94% agreement between EPA method 1600 and Enterolert. The beach management decision agreement between E. coli QPCR and Colilert-18 was 94%. The samples showing disagreement suggested an underestimation bias for QPCR

Trends in total Vibrio spp. and Vibrio vulnificus concentrations in the eutrophic Neuse River Estuary, North Carolina, during storm events

Vibrio spp. are ubiquitous members of aquatic microbial food webs that can be pathogenic to humans and a range of other organisms. Previously published predictive models for Vibrio spp. concentrations in estuarine and coastal waters, based only on salinity and temperature, are 70 to 75% accurate during 'normal' conditions (e.g. not during storms or drought). We have conducted a preliminary comparison of the output from this type of model to the natural concentrations of both total Vibrio spp. and the potentially pathogenic Vibrio vulnificus when measured during tropical storms. Water samples were collected in situ from a deployed platform in the Neuse River Estuary (NRE), North Carolina, USA, during 2 storm events: Hurricane Ophelia and Tropical Storm Ernesto. Total Vibrio spp. concentrations were measured using culture-based methods and V vulnificus levels were determined using a newly developed, rapid quantitative polymerase chain reaction (QPCR) assay. Results were analyzed in relation to environmental parameters and to concentrations of the fecal indicator bacteria Escherichia coli (EC) and Enterococcus spp. (ENT). Total concentrations of Vibrio spp. in the NRE were often orders of magnitude higher than those predicted by a previously published model. These large deviations from model predictions may indicate contributions from storm forcing (e.g. resuspension, surges) that are missing from the calm weather observations used to build these models

Participant written evaluation responses focused on the workshop environment.

<p>Q5: The workshop was well organized; Q6: The atmosphere of the workshop was professional; Q7: The lodging arrangements were clean and appropriate for this venue; Q8: The food selection was appropriate, on time, and enjoyable; Q9: Transportation during the workshop was on time, comfortable, and drivers were courteous.</p