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Published in final edited form as: J Environ Manage. 2024 Oct 3;370:122711. doi: 10.1016/j.jenvman.2024.122711

Comparison of liquid and filter sampling techniques for recovery of Bacillus spores and Escherichia coli from environmental water

Ahmed Abdel-Hady a, Mariela Monge b, Denise Aslett a, Anne Mikelonis c, Abderrahmane Touati a, Katherine Ratliff c,*
PMCID: PMC11836889  NIHMSID: NIHMS2054413  PMID: 39366227

Abstract

Historically, detecting water contamination has involved collecting and directly analyzing liquid samples, but recent advances in filter sampling methods offer numerous potential advantages. Emerging technologies, including environmental DNA (eDNA) samplers, could be used for remote microbial contamination sampling, but work is needed to determine if target microorganisms can be recovered from filters at comparable levels to traditional sampling methods. In this study, Escherichia coli and a surrogate for Bacillus anthracis spores were sampled from synthetic stormwater and quantified using both direct liquid and filter methods, and dwell time tests compared microorganism persistence in water and on filters. At nearly all tested timepoints, the recoveries of spores from membrane filters were within 0.5 log10 colony forming units per sample (CFU/sample) compared to the liquid-only samples, suggesting that the use of filter sampling is a feasible alternative to liquid-based sampling, and samples were held for up to 4 weeks without significant sample degradation. Recoveries for E. coli remained relatively consistent for ~3 days in phosphate buffered saline (PBS), in synthetic stormwater, and on membrane filters, but decreases in recoveries were observed for samples held for >3 days. These results indicate that emerging water sampling technologies, which reduce logistical burdens and offer potential cost savings, can be leveraged to characterize biological contamination in water matrices with multiple types of microbiological agents.

Keywords: eDNA, Filter sampling, Escherichia coli, Bacillus anthracis, Bacillus atrophaeus var. globigii

1. Introduction

Microbiological monitoring of different water matrices has many uses and plays a key role in maintaining public health. In the United States alone, wastewater treatment facilities process approximately 34 billion gallons of wastewater each day, which requires the removal of various contaminants, including enteric bacteria, before discharging from Publicly Owned Treatment Works (POTW) (Oliver et al., 2005; U.S. EPA, 2022). Historically, fecal indicator bacteria (FIB) such as Escherichia coli (E. coli) have been used to assess the sanitary quality in multiple water sources including drinking water, wastewater, irrigation water, recreational waters, and rivers (Ishii and Sadowsky, 2008; Odonkor and Ampofo, 2013; Tyrrel et al., 2006). Although it is often considered a poor surrogate for some pathogenic bacteria, viruses, and protozoa, E. coli is still widely used as an important indicator for fecal contamination, and its presence can signal that a contamination event may have occurred, suggesting that more sampling is needed to find the source (Brookes et al., 2005; Odonkor and Mahami, 2020).

In addition to the unintentional spread of contamination, the intentional contamination of environmental waters also poses a significant threat to human health., and characterizing the extent of outdoor contamination following a bioterrorism event has been identified as a knowledge gap (Calfee et al., 2017). For example, Bacillus anthracis (Ba) spores could pose a threat to drinking water and irrigation systems through direct injections or by contamination of groundwater supplies by stormwater runoff following a wide-area outdoor release (Lindquist et al., 2007; Mikelonis et al., 2021). Recently, various water sampling techniques including collecting and quantifying concentrations of viral RNA have been leveraged to monitor emerging pathogens such as SARS-CoV-2 in wastewater (D’Aoust et al., 2021; Feng et al., 2021; Juel et al., 2021). For example, Moumanis et al. recently evaluated the use of a semi-automated water sampling module to sample for L. pneumophila (Moumanis et al., 2021). Hassen et al. evaluated the same water sampling module to sample for B. thuringiensis and B. cereus spores (Hassen et al., 2023). The U.S. Environmental Protection Agency (EPA) has also evaluated different methods of concentrating water samples in order to take a representative sample of large volumes of water where the concentration of biological agents may be low. Axial flow hollow-fiber ultrafiltration has been used to concentrate large volume water samples (e. g., up to 100 L) to detect various bacterial and viral surrogates including surrogates for Ba (Bacillus anthracis Sterne and Bacillus atrophaeus var. globigii) and viral surrogates (bacteriophages MS2, PhiX174, and Phi8) (Gallardo et al., 2019; Humrighouse et al., 2015; Rhodes et al., 2011), demonstrating the applicability of concentrating large water samples into more manageable volumes for analysis and increased detection rates.

Traditional water sampling typically includes collecting large volumes of water and shipping them to laboratories for analysis, sometimes with analysis hold times as short as 8 h. Current recommendations from the EPA and the American Public Health Association include sampling a minimum of 100 mL and up to 1 L of water for analysis for each collected sample (Rice et al., 2012; U.S. EPA, 2002). A potential disadvantage of traditional sampling is the need for personnel to be present at the site of collection, which can lead to limitations in sampling locations at a contaminated site (to ensure safety of workers), reduced laboratory response times, and potential exposure of sampling personnel to pathogens. Additionally, grab sampling or spot sampling may not support holistic characterization of contamination due to the failure to detect episodic contamination, yet it remains expensive and labor-intensive (Madrid and Zayas, 2007). After collection, large volumes of potentially contaminated water need to be transported to analytical laboratories, resulting in large shipping costs and other logistical problems regarding regulations for shipping hazardous substances, including potentially expensive shipping containers, sample storage issues, short required hold times for sample analysis, and increased costs associated with expedited sample processing. Additionally, samples are typically shipped and stored at cold temperatures, because refrigeration is a deterrent to bacterial decomposition, cell division, growth, and death (Brozel and Cloete, 1991), but cold storage can be a challenge to maintain due to limitations with equipment, space, and power (particularly following disasters). Moreover, cold storage may be prohibitively challenging or expensive to maintain at the point of collection if there is a delay between when a sample was collected and when it is shipped to a lab, which is often not immediate.

Automated sampling technologies, such as the ISCO autosampler (Teledyne ISCO, Lincoln, NE USA), which are portable sampling devices that can collect sequential or composite liquid samples, can be deployed for remote collection of water samples, and they can also alleviate some of the concerns over discrete sampling and the use of in person sampling (Hallett Sascha et al., 2012). Automated samplers are especially useful when sampling areas are highly contaminated or otherwise too dangerous for personnel and when samples can be retrieved after some period of time. However, large volumes of potentially contaminated water would still need to be transported for analysis. One potential drawback to the use of automated samplers is sample integrity and potential “bottle effects” (Ghazaleh et al., 2014). Bottle effects have been defined as changes in genetic, biochemical, and physical aspects of a sample after enclosure (Madsen, 2015). Some studies hypothesize a “safe period” of less than 24 h in which to analyze samples, and several studies indicate that analysis should be completed as soon as possible to minimize “bottle effects” and accurately detect the microorganisms present in the sample (Ghazaleh et al., 2014). Another potential drawback from using automated samplers would be potential cross contamination; however, engineering controls (e.g., additional automated flushes of the sampling system) could be designed to minimize this issue. Purges, shorter sampling lines and increasing flow rates are currently used to help prevent potential cross contamination.

Recently, environmental DNA (eDNA) sampling methods have gained popularity for the surveillance and detection of rare and invasive species (Bowers et al., 2021; Liang and Keeley, 2013; Thomas et al., 2018). These technologies could be used to collect samples from various water matrices potentially contaminated with other biological matter, including FIB or spores. Examples of various eDNA samplers include the Smith-Root eDNA sampler (Smith-Root, Vancouver, WA, USA), a modified Hess sampler (Sepulveda et al., 2019), and a tow net connected to a dolphin bucket with mesh filters embedded (Schabacker et al., 2020). Additionally, researchers at Oregon State University have developed a remotely deployable automated sampling unit which uses in-line filters (Nguyen et al., 2019). These devices are used to capture discrete water samples and filter them over individual membranes. After a sampling event, the membranes are collected from the samplers, transported to a processing laboratory, and typically analyzed to determine the presence of specific target DNA; however, the presence of DNA does not confirm the presence of an infectious microorganism. If these sampling techniques also allow for enumerating viable target organisms, then this could provide additional valuable information for determining exposure risks and better protecting public health. The utilization of filters at the site of collection would reduce the burden of transporting large volumes of liquid samples and could be leveraged to sample larger volumes of water at various timepoints to provide more comprehensive information regarding water quality or the extent of contamination. Additionally, the use of on-site filters could increase the use of drone-assisted sampling by decreasing the payloads, thereby allowing collection of an increased number of samples from multiple locations (Lally et al., 2019). However, knowledge gaps remain regarding the comparability of these emerging techniques compared to traditional sampling methods. To leverage these technologies for the detection of FIB and spores and to understand the timing constraints surrounding sample collection and analysis, research is needed to verify the collection, survival, and persistence of target organisms on various membrane materials used to filter different types of liquid matrices.

The purpose of this study was to compare the sampling efficiency and persistence of Bacillus atrophaeus var. globigii (Bg), a surrogate for Ba, and E. coli, a common FIB, collected by filtration with commercially available membrane filters from various liquid matrices to direct analysis of those liquids. Both of these bacteria are associated with a known public health concern. Comparing recoveries from samples analyzed directly from biologically contaminated water to filters that have been used to collect samples from the same water is necessary to demonstrate the feasibility of this method and the adaption of emerging filtration-based sampling equipment like eDNA samplers for quantifying bacterial contamination in environmental water. Moreover, an improved understanding of sample hold times and necessary conditions for storage informs the development of practical, reliable sampling methods that can be used for a variety of public health purposes. Previous studies have evaluated the persistence of Bg in clean liquid matrices (sterile deionized water) and on cellulose nitrate filters (Ratliff et al., 2023). This study expands the prior research by evaluating the persistence of Bg using a synthetic stormwater liquid matrix with sample hold times up to four weeks under ambient conditions. The persistence of E. coli was also evaluated using a clean liquid matrix and with synthetic stormwater with samples held at 4 °C or ambient temperatures. Stormwater was chosen as the additional experimental liquid matrix for this work because of its potential to pose an exposure risk to humans in populated urban areas following a bioterrorism incident (Mikelonis et al., 2021; Mikelonis et al., 2020), and high concentrations of FIB are also associated with urban stormwater runoff (Paule-Mercado et al., 2016).

2. Materials and methods

2.1. Biological agents and analyses

Bacillus atrophaeus var. globigii (Bg) and Escherichia coli (E. coli) were the biological agents used in this study. Bg is a nonpathogenic spore forming bacterium and has been used historically in biodefense research as simulant for Ba, the causative agent of anthrax (Gibbons et al., 2011). Bg spores were obtained from the United States Army DEVCOM Chemical Biological Center, Edgewood, MD (prepared 5/17/2021). The spore inoculum quality was evaluated by titer enumeration following heat shock treatment at 80 °C for 20 min to ensure that the vegetative cell component was <5–10%. E. coli is a Gram-negative bacterium, which is naturally occurring in the intestines of mammals, and has long served as a fecal waste indicator in water (Odonkor and Ampofo, 2013). E. coli was obtained from the American Type Culture Collection (ATCC # 15597, Manassas, VA).

Bg inocula were prepared in phosphate buffered saline (PBS, P0196, Teknova, Hollister, CA) with 0.05% Tween 20 (PBST, P1191, Teknova, Hollister, CA). E. coli inoculums were prepared from an overnight live culture, grown in LB broth (Catalog number DF0446-07-5, Thermo Fisher Scientific, Waltham, MA, USA) and diluted in PBS. Prior to dilution, the optical density of the culture was measured at 600 nm using a spectrophotometer (Genesys 20, Thermo Fisher Scientific). Two inoculum concentrations (1 × 108 CFU/mL and 1 × 105 CFU/mL) were prepared for each microorganism such that 100 μL inoculations provided target concentrations of 1 × 107 CFU/sample and 1 × 104 CFU/sample, respectively. Inoculum titer checks were performed with each test.

Bg samples were plated on tryptic soy agar (TSA) plates (Catalog number DF0369-17-6, Thermo Fisher Scientific) and E. coli samples were plated on Luria-Bertani agar (LB) (Catalog number DF0445-17-4, Thermo Fisher Scientific). All plating was performing in triplicate using either spiral plating (easySpiral Pro, InterScience Laboratories, Woburn, MA, USA), manual spread plating, or filter plating with sterile 0.45 μM MicroFunnel™ filter funnel (item #4852 [for Bg] or #4805 [for E. coli], PALL, Port Washington, NY, USA). All samples were incubated for 18–24 h at 35 °C ± 2 °C prior to enumeration. Filter plating of samples decreased the limit of detection for all samples to approximately 1–2 CFUs per sample.

2.2. Filtration media and sampling methods

Based on the results of prior extraction efficiency tests using various filter materials (Ratliff et al., 2023), Bg experiments were conducted using the filter material with the highest measured sampling efficiency, 0.45 μM cellulose nitrate filters (product number DS0205–4045, Thermo Fisher Scientific), and E. coli experiments were conducted using 0.45 μM cellulose acetate filters (product number DS0210–4045, Thermo Fisher Scientific). As a first step in this research, water filtration was performed using simple filtration units to minimize losses that may occur with more lengthy tubing or complex equipment (and to minimize the risk of cross-contamination); filters were properly seated within the Nalgene Reusable Filter Units (product number 300–4050, Thermo Fisher Scientific), which were connected to a house vacuum line to provide negative pressure. The filter units were sterilized by autoclaving (SV 120 scientific pre-vacuum sterilizer; STERIS Amsco, Mentor, OH, USA) prior to each use.

Test samples were prepared in either filter-sterilized deionized (DI) water, filter-sterilized PBS (PBS), or synthetic stormwater. Synthetic stormwater was prepared according to Mikelonis et al. (2020) with zinc (catalog number ICP-MS-70N-0.1X-1) and copper (catalog number ICP-MS-15N-0.1X-1, Accustandard Inc., New Haven, CT, USA) inductively coupled plasma mass spectrometry standards used in place of the zinc sulfate heptahydrate and copper sulfate pentahydrate, respectively (zinc and copper are metals commonly found in urban stormwater matrices). PBS was used with E. coli as a clean liquid matrix. Filtration of both Bg and E. coli was tested using the synthetic stormwater. Liquid matrix aliquots of 100 mL were inoculated with 100 μL of the respective inoculums, and samples were then filtered through the filter funnels containing the appropriate filter membrane material. The 100 mL aliquots were prepared to achieve either a high titer (107 CFU/sample) or low titer (104 CFU/sample) for both microorganisms. These values were chosen to be consistent with observed environmental FIB concentrations (Paule-Mercado et al., 2016) and are consistent with previous water filter sampling research using spores (Ratliff et al., 2023). After filtration of the samples, funnels were rinsed with sterile DI water, and the filters were aseptically transferred to 50-mL conical tubes and either eluted and analyzed immediately or held in the closed tubes at either 4 °C or ambient temperatures (E. coli only) for prescribed time periods (0 days, 1 day, 3 days, 1 week, 2 weeks, and 4 weeks) as potential representative sample storage periods between collection and analysis. Given their more persistent nature as spores compared to vegetative bacteria, Bg was held up to 4 weeks, but E. coli was only held up to two weeks. After the hold times had lapsed, bacteria and spores were eluted from the filters by adding 20 mL of PBST (Bg) or PBS (E. coli) to each tube and vortexing continuously for 2 min using a multi-tube vortexer (VX-2500, VWR International, LLC., Radnor, PA, USA) set to its maximum setting. Positive controls consisted of only the inoculated liquid matrix without the filtration step (i.e., inoculated suspensions stored under the same conditions as the filter samples) at each time point. All samples were analyzed in triplicate.

2.3. Statistics

Single factor ANOVA was conducted using the Data Analysis ToolPak for Microsoft Excel (for Microsoft 365 Version 2108) to compare recoveries between sample types (enumerating directly from liquid samples or samples that had been filtered and eluted) and recoveries over time. Statistical significance was determined at the α = 0.05 level.

3. Results and discussion

3.1. Bacillus atrophaeus var. globigii (Bg) results

The recoveries for Bg comparing the filter test samples and the positive control liquid samples for both the high and low titers in synthetic stormwater are shown in Fig. 1. For the high titer samples, recoveries for the liquid based positive controls were observed to slightly decline from 7.6 ± 0.02 CFU/sample on Day 0 to a 7.0 ± 0.03 CFU/sample after 4 weeks. A similar trend was observed for the high titer filter samples, which exhibited recoveries ranging from 7.5 ± 0.02 CFU/sample on Day 0–7.1 ± 0.03 CFU/sample at 4 weeks. ANOVA comparing the positive control recoveries to the filter test sample recoveries shows no statistically significant difference (p = 0.755).

Fig. 1.

Fig. 1.

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Recovery of Bacillus atrophaeus var. globigii spores at high (~107 CFU/sample) and low (~104 CFU/sample) titers sampled directly from synthetic stormwater or from stormwater filtered through cellulose nitrate filters after specified sample hold times.

Similarly for the low titer samples, overall recoveries for both the liquid-based positive controls and the filter test samples showed a slight declining trend. However, the filter test sample recoveries on Day 1 and Week 1 are lower than the recoveries for Weeks 2 and 4. The lower-than-expected recoveries on Day 1 and Week 1 filtered samples may have been a result of inherent variances in microbiological samples or potential variability during the inoculation, extraction, or analytical processes. These outliers may have contributed to significant differences in the two data sets. ANOVA for the low titer data sets showed that there was a statistically significant (p = 0.047) difference between the positive controls and the filter test samples. However, for the longest and perhaps most important time points of 2 weeks and 4 weeks, there were no statistically significant differences in the two data sets (p = 0.340). Similar recoveries at these longer hold times demonstrate that a great flexibility in the transport and analysis of samples is possible while still meeting sampling needs, which could allow for expanded capabilities (e. g., sampling area to be widened or more frequently sampled for more extensive characterization). Overall, spore recovery on the membrane filters was typically within 0.1 log10 CFU/sample for the high titer samples and 0.5 log10 CFU/sample for the low titer samples, suggesting that the filter membrane can be used to characterize spore content within an order of magnitude when used to sample from contaminated stormwater rather than analyzing the stormwater directly under these conditions. These findings are consistent with Ratliff et al. (2023), which demonstrated relatively high spore recoveries from cellulose nitrate filters when sampled from deionized water and consistent recoveries with sample hold times up to four weeks. This work expands upon those findings by demonstrating that these sampling techniques are similarly viable for sampling spores in more environmentally relevant waters.

3.2. Escherichia coli results

Recoveries for all E. coli tests, comparing recoveries from clean (PBS) and stormwater matrices, are shown in Fig. 2. A direct comparison between samples held at ambient and at 4 °C was also performed to help inform decision makers on the feasibility of using remote samplers in areas where the samples may not be retrieved immediately and exposed to various climate conditions. These temperature comparison experiments were conducted with E. coli (instead of Bg) to determine holding time limitations with vegetative bacteria, which are generally more susceptible to degradation than bacterial spores (Spaulding and Emmons, 1958), and therefore represent a greater challenge for using this potential sampling technique. Generally, recoveries for all E. coli samples decreased over time, regardless of titer, with the largest decrease in recovery occurring between weeks 1 and 2. The exception to this was both the high and low titer positive controls in synthetic stormwater held at 4 °C. These results suggest that the synthetic stormwater may have some protective effect on the degradation and/or death of the E. coli cells compared to cleaner matrices, especially when held at 4 °C. Additionally, the synthetic stormwater filter membrane samples held at 4 °C displayed a smaller difference in CFU recovered over time compared to the PBS filter samples held at 4 °C or the synthetic stormwater filter samples held at ambient conditions. These results suggest that not only is the storage temperature of samples important for sample preservation, but the liquid matrix also has a significant impact on cell viability (p = 0.0016). Although there were significant differences between the CFU recovered on the filters compared to the liquid matrices, especially after 3 days, the use of filter sampling may still provide a reasonable way to estimate relative levels of contamination within selected waters and could be leveraged to sample larger volumes at more locations than would be feasible if analyzing the liquids directly. Overall, this approach may help increase the sensitivity of water sampling by increasing the volume of water and/or temporal frequency that is sampled by the filters and potentially eliminate the transport and analysis of large sample volumes often associated with traditional sampling methods.

Fig. 2.

Fig. 2.

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Recovery of E. coli from positive control (liquid samples that did not undergo a filtration step) and filter test samples from phosphate buffered saline (PBS) and synthetic stormwater at A) high (~107 CFU/sample) and B) low (~104 CFU/sample) titers after specified hold times at 4 °C or ambient conditions.

Standard methods for quantifying coliforms using membrane filtration techniques (Standard Methods Committee, 2022) call for the filters to be directly enumerated by culturing on the filters themselves, but here we show that FIB can be recovered from filtered samples following an elution step, which may help to overcome challenges cited in the standard methods relating to quantifying high microorganism counts. These methods also cite high turbidity and the presence of toxic compounds as potential interferences to quantifying coliform concentrations directly on filters, which may also be limitations to the methods demonstrated here, although the presence of the metals used in this work (zinc and copper, both of which have demonstrated antimicrobial properties in water (Pasquet et al., 2014; Vincent et al., 2016)), did not prohibit recovery of E. coli from the stormwater matrix tested here. Additional research is needed to constrain the limitations of these methods in different water compositions.

4. Conclusions

The results shown here demonstrate the potential feasibility and applicability of using filter sampling to replace or reduce traditional liquid sampling for target microbiological agents in potentially contaminated water matrices. Both Bg and E. coli were successfully recovered from liquid samples using filter membranes and remained viable for 4 and 2 weeks, respectively. The temperature at which samples were held and the constituents of the various liquid media had a significant impact on the viability of E. coli over time, with up to a log difference observed between recoveries. These results highlight the need to optimize collection methods based on microbiological targets, the environmental conditions from which they are sampled, and the means of sample collection and storage prior to analysis. Future work is needed to evaluate filter sampling in larger field scale studies, including other microbiological agents of interest and various preservation medias.

Acknowledgements

The authors gratefully acknowledge EPA contractors Brian Ford, Rachael Baartmans, and Lesley Mendez Sandoval for their support in conducting the research, Ariamalar Selvakumar and Josh Steenbock for internal technical reviews of this manuscript, and Ramona Sherman for quality assurance support.

Footnotes

Disclaimer

The EPA, through its Office of Research and Development, directed the research described herein conducted through contract 68HERC20D0018 with Jacobs Technology, Inc. It has been subjected to the Agency’s review and has been approved for publication. Mention of trade names, products or services does not convey official EPA approval, endorsement, or recommendation.

CRediT authorship contribution statement

Ahmed Abdel-Hady: Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation. Mariela Monge: Writing – review & editing, Visualization, Methodology, Investigation, Formal analysis, Data curation. Denise Aslett: Writing – review & editing, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation. Anne Mikelonis: Writing – review & editing, Investigation, Conceptualization. Abderrahmane Touati: Writing – review & editing, Supervision. Katherine Ratliff: Writing – review & editing, Supervision, Resources, Project administration, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data that support the findings of this study are available at https://doi.org/10.23719/1528788.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data that support the findings of this study are available at https://doi.org/10.23719/1528788.

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