Geology Reports⌕ Search
Home/Archive/USGS

USGS · 70223149

Evaluation of a modified rapid viability-polymerase chain reaction method for Bacillus atrophaeus spores in water matrices

Rebecca N. Bushon·Amie M.G. Brady·Christopher M. Kephart·Vicente Gallardo
View original source ↗Export metadata ↓

Abstract

A rapid method that provides information on the viability of organisms is needed to protect public health and ensure that remediation efforts following a release of a biological agent are effective. The rapid viability-polymerase chain reaction (RV-PCR) method combines broth culture and molecular methods to provide results on whether viable organisms are present in less than 15 h. In this study, a modified RV-PCR (mRV-PCR) method was compared to a membrane-filtration culture method for the detection of viable Bacillus spores in water matrices. Samples included small and large volumes of chlorine and non‑chlorine treated tap water. Large volume water samples (up to 100 L), were processed by ultrafiltration using a semi-automated waterborne pathogen concentrator, followed by centrifugation as a secondary concentration technique. The concentrated samples were analyzed by mRV-PCR and culture methods. The overall agreement between the mRV-PCR and culture methods when seed concentrations were greater than 10 spores per sample volume analyzed was 96%. The total time from the start of sample processing to the final sample result for the mRV-PCR method was decreased by approximately 2 h, in comparison to the previously published RV-PCR method because of the incorporation of shorter, more efficient primary and secondary concentration steps and a shorter DNA extraction technique. Overall, this study confirmed that RV-PCR is a promising approach for identifying viable Bacillus spores in small- and large-volume water samples and for producing results in less time than traditional culture methods.

Explore related subjects

Journal of Microbiological MethodsArticle

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Rebecca N. Bushon, Amie M.G. Brady, Christopher M. Kephart, Vicente Gallardo. 2021. Evaluation of a modified rapid viability-polymerase chain reaction method for Bacillus atrophaeus spores in water matrices. https://doi.org/10.1016/j.mimet.2021.106293

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related USGS reports

USGS2020

Validation of a screening method for the detection of colistin-resistant E. coli containing mcr-1 in feral swine feces

A method was developed and validated for the detection of colistin-resistant Escherichia coli containing mcr-1 in the feces of feral swine. Following optimization of an enrichment method using EC broth supplemented with colistin (1 µg/mL) and vancomycin (8 µg/mL), aliquots derived from 100 feral swine fecal samples were spiked with of one of five different mcr-1 positive E. coli strains (between 100 and 104 CFU/g), for a total of 1,110 samples tested. Enrichments were then screened using a simple boil-prep and a previously developed real-time PCR assay for mcr-1 detection. The sensitivity of the method was determined in swine feces, with mcr-1 E. coli inoculums of 0.1-9.99 CFU/g (n = 340), 10-49.99 CFU/g (n = 170), 50-99 CFU/g (n = 255), 100-149 CFU/g (n = 60), and 200-2,200 CFU/g (n = 175), which were detected with 32%, 72%, 88%, 95%, and 98% accuracy, respectively. Uninoculated controls (n = 100) were negative for mcr-1 following enrichment.

Journal of Microbiological Methods↗
USGS2020

Nowcasting methods for determining microbiological water quality at recreational beaches and drinking-water source waters

Nowcasts are tools used to provide timely and accurate water-quality assessments of threats to drinking-water and recreational resources from fecal contamination or cyanobacterial harmful algal blooms. They use mathematical models and techniques to provide near-real-time estimates of fecal-indicator bacteria (FIB) and cyanotoxin concentrations. Techniques include logic-based thresholds, decision trees (built with machine learning), multiple linear and binary logistic regression, artificial neural networks, and process-based deterministic models. The type of site (freshwater, marine, or river) and dependent variable (FIB or cyanotoxin) dictate which explanatory variables are used in models. Nowcast systems notify the public of associated public-health risks and can also be used to manage data for FIB models; work is ongoing to incorporate cyanotoxin models into some nowcasts. The Great Lakes NowCast in the USA has been operational since 2010 and includes 25 lake beaches and one recreational river site. Examples of other operational FIB nowcasts are described for locations in the USA and around the world. In many cases, models predicted exceedances of FIB standards with accuracies as good as or better than using the previous measured FIB concentration (persistence method). Accuracy and timeliness are vital to beach management decisions that protect public health and support the local recreation-driven economy. Nowcasts benefit the public by providing estimates of water-quality conditions in near-real-time. Managers can use nowcasts at recreational and drinking-water treatment plant sites when FIB or cyanotoxins are projected to be elevated to target sample collection, to provide near-real-time recreational advisories to the public, or to preemptively optimize drinking-water treatments or change intake options to mitigate possible adverse effects on drinking-water quality.

Journal of Microbiological Methods↗
USGS2020

Utilization of multiple microbial tools to evaluate efficacy of restoration strategies to improve recreational water quality at a Lake Michigan Beach (Racine, WI)

Hydro-meteorological conditions facilitate transport of fecal indicator bacteria (FIB) to the nearshore environment, affecting recreational water quality. North Beach (Racine, Wisconsin, United States), is an exemplar public beach site along Lake Michigan, where precipitation-mediated surface runoff, wave encroachment, stormwater and tributary outflow were demonstrated to contribute to beach advisories. Multiple restoration actions, including installation of a stormwater retention wetland, were successfully deployed to improve recreational water quality. Implementation of molecular methods (e.g. human microbial source tracking markers and Escherichia coli ( E. coli ) qPCR) assisted in identifying potential pollution sources and improving public health response time. However, periodic water quality failures still occur. As local beach managers reassess restoration measures in response to climatic changes, use of expanded microbial methods (including bacterial community profiling) may contribute to a better understanding of these dynamic environments. In this 2-year study (2015 and 2019), nearshore/offshore Lake Michigan, stormwater, and tributary samples were collected to determine if, 1) the constructed wetland (~50 m from the shoreline) continued to provide stormwater separation/retention and 2) mixing between onshore sources, Root River and Lake Michigan, was increasing due to rising precipitation/lake levels. Monthly rainfall totals were 1.5× higher in 2019 than 2015, coinciding with a 0.63 m lake-level rise. The prevalence of more intense, onshore winds also increased, facilitating interaction between potential reservoirs of FIB with nearshore water through wind driven waves and lake intrusion, e.g. beach sands and the adjacent Root River. While a strong relationship existed between wet weather wetland and North Beach nearshore E. coli concentrations (all sites), bacterial communities were strikingly different. Conversely, bacterial community overlap existed between the Root River mouth and nearshore/offshore sites. These results suggest the constructed wetland can accommodate the climate-related changes observed in this study. Future restoration activities could be directed towards upstream tributary sources in order to minimize microbial contaminants entering Lake Michigan.

Wisconsin↗