Culturable E. coli as Surrogate for Culturable V. cholerae in Surface Disinfection Testing with Chlorine
Publication: Journal of Environmental Engineering
Volume 147, Issue 4
Abstract
During outbreaks, contaminated surfaces can contribute to the spread of cholera; as such, surface disinfection with chlorine is widely recommended. However, confirmation of surface disinfection efficacy against Vibrio cholerae requires testing for the V. cholerae bacteria, which is not commonly completed in outbreak settings. We investigated whether culturable Escherichia coli (commonly measured in outbreak settings) is an appropriate surrogate for culturable V. cholerae by conducting experiments using 0.02% chlorine made with sodium dichloroisocyanurate (NaDCC) on six nonporous and porous surfaces. Across all test conditions, culturable E. coli were reduced by 1.3 to and culturable V. cholerae by 4.9 to 7.0 log. Results suggest culturable E. coli is a conservative surrogate for culturable V. cholerae, and it is recommended that E. coli can be used in households and health care facilities in cholera outbreaks as a conservative indicator of adequate disinfection. Further research is recommended on the relative importance of fomite transmission in cholera contexts, the role that viable but nonculturable (VBNC) V. cholerae (not tested herein) might have in transmission, and potential surrogates for VBNC V. cholerae.
Introduction
Infection by Vibrio cholerae bacteria can cause severe dehydration through excessive vomiting and diarrhea, which can be lethal within hours if untreated (Sack et al. 2004). In 2017, over 1.2 million cholera cases and 5,600 deaths were reported globally (WHO 2018). However, modeling studies estimate the annual global disease burden is much greater, at 2.9 million cases and 95,000 deaths (Ali et al. 2015). Cholera primarily affects areas that lack adequate water and sanitation infrastructure, and household contacts of patients have an approximately 100 times increased risk of contracting cholera compared with the general public (George et al. 2018). Human-to-human transmission (e.g., via shared contaminated food or water) is estimated to account for 41%–95% of disease transmission in outbreaks (Mukandavire et al. 2011). Indirect cholera transmission through exposure to contaminated surfaces or objects (fomites) has an associated infection risk as high as 8.2% [95% confidence interval (CI) 2.1%–27.1%] over 11 days (Sugimoto et al. 2014), and is thus a potentially important transmission pathway. The infectious dose of V. cholerae ranges from to cells in the absence of a buffer to neutralize stomach acid, and from to cells when a neutralizing buffer is present; however, a higher infectivity of ingested cells can lower the infectious dose (Nelson et al. 2009).
Disease transmissibility via fomites is influenced by a pathogen’s persistence on surfaces and resistance to disinfection (Tuladhar et al. 2012). Surface disinfection with chlorine is widely recommended in cholera treatment centers and patient households during outbreaks (CDC 2018; Olson et al. 2017; UNICEF, n.d.). Few studies, however, have evaluated surface disinfection of V. cholerae with chlorine compounds. One study tested (a nonstandard concentration) pH-adjusted bleach (sodium hypochlorite and acetic acid) sprayed on aluminum, glass, wood, carpet, and concrete surfaces (Calfee and Wendling 2013). Culturable V. cholerae recovery on wood and concrete were too low to assess disinfection efficacy, while log reduction values (LRV) on aluminum, glass, and carpet were 5 to after 15–30 min of exposure. Additional testing with 0.2% chlorine solutions, which are more representative of those used on surfaces in outbreak settings, found LRV after 10 min on nonporous surfaces, but lower LRVs and greater variability on porous surfaces (String et al. 2020). This research further informs surface disinfection practices against V. cholerae in outbreak settings.
Surrogate organisms are often used to evaluate disinfection practices for pathogenic or difficult-to-culture organisms such as V. cholerae. Surrogates are ideally simple and inexpensive to test, and slightly more resistant to disinfection than the pathogen of interest (Sinclair et al. 2012). To identify potential surrogates for V. cholerae, we performed a literature review and found two relevant studies in which culturable Escherichia coli and culturable V. cholerae were compared in suspension tests, with results suggesting that E. coli is slightly more resistant to chlorine disinfection than V. cholerae (Adebisi et al. 2017; Jones et al. 1992). Both organisms have also demonstrated sensitivity to protein aggregation when exposed to chlorine (Winter et al. 2008), suggesting that they may undergo similar inactivation mechanisms. E. coli is easier and safer to culture than V. cholerae, making it a good candidate for surrogacy testing. We therefore sought to determine if culturable E. coli could be an appropriate surrogate for culturable V. cholerae in disinfection efficacy tests on nonporous and porous surfaces; we hypothesized that E. coli could potentially be an appropriate surrogate.
Materials and Methods
Surface Carrier Selection and Preparation
We selected six materials representative of surfaces in low-income health facilities and homes: stainless steel (medical instruments); heavy-duty tarp (floors/walls of health facilities); nitrile (personal protective equipment); glazed ceramic (plates/tableware); wood (doors, furniture); and foam (mattresses).
Nonporous surfaces were 8-cm-diameter discs of type 430 brushed stainless steel (McMaster Carr, Elmhurst, Illinois), heavy-duty tarp (Amazon.com; Direct Tarp, Seattle, Washington State), and nitrile (Amazon.com; Small Parts, Seattle, Washington State), and 10-cm-diameter glazed ceramic plates (The Home Depot, Georgia, US). Porous surface carriers were squares of polyurethane foam (McMaster Carr, Elmhurst, Illinois) and wood (The Home Depot, Albany, Georgia). Before each test, stainless steel, wood, ceramic, and foam carriers were sterilized by autoclaving at 121°C and 15 kPa for 15 min. Heavy-duty tarp and nitrile discs were soaked in 70% ethanol overnight, rinsed with sterile water, and dried using a sterile surgical towel.
Disinfectant Selection and Preparation
The World Health Organization (WHO) recommends the use of chlorine disinfectants in V. cholerae outbreaks (WASH Working Group 2019). Four chlorine compounds constitute the majority of disinfectants used in low-resource outbreak settings: sodium hypochlorite (NaOCl), sodium dichloroisocyanurate (NaDCC), high-test calcium hypochlorite (HTH), and electrolyzed water on site (gNaOCl). Previous work demonstrated no significant differences between these compounds in surface disinfection of E. coli and further study of NaOCl, NaDCC, and HTH found similar surface disinfection efficacy against V. cholerae (Gallandat et al. 2017; String et al. 2020). Both previous studies were conducted under testing conditions similar to the conditions described herein with chlorine concentrations of 0.2%–0.5%. Thus, we prepared chlorine solutions using NaDCC as a representative chlorine disinfectant for this testing. Pretesting was conducted with E. coli on stainless steel using 0.002%–0.1% chlorine solutions to determine which concentration would provide residual bacteria in the countable range [1–250 colony forming units (cfu) per plate] after exposure times of 1, 5, and 10 min. Based on pretesting, a concentration of 0.02% chlorine (200 mg chlorine/L) made with NaDCC was selected, which was expected to allow comparisons between E. coli and V. cholerae inactivation rates over 1–10 min.
On each test day, NaDCC chlorine solutions were prepared using Aquatabs granules with 50% active chlorine (Medentech, Ireland) dissolved in Milli-Q water. Chlorine concentration was then confirmed to be within 10% of the target by iodometric titration (Method #8209, Hach Company, Loveland, Colorado), and used for disinfection within 3 h to ensure stability (Iqbal et al. 2016).
Test Organisms
One day prior to testing, cultures of E. coli (ATCC 25922) and V. cholerae (El Tor N16961, ATCC 39315, biosafety level 2) were prepared from a single streak plate colony in Luria-Bertani (LB, BD Difco, Franklin Lakes, New Jersey) broth and tryptic soy broth (TSB, BD Difco, Franklin Lakes, New Jersey), respectively, and incubated at 35°C overnight. The culture was then diluted in broth and incubated for 2.5 h. Cell density was estimated by spectrophotometry (GeneQuant100, GE Healthcare Life Sciences, UK) and cultures with at least were used to inoculate surface carriers. Each test day, the inoculation cultures were processed as described in the section “E. coli and V. cholerae Sample Processing and Data Analysis.”
Disinfection Experiments
Surface disinfection efficacy was evaluated following methods adapted from the ASTM International Quantitative Carrier Test Method and described elsewhere (Gallandat et al. 2017). Surface carriers were placed in Petri dishes, inoculated with 2 mL bacterial culture, and left to dry in a biosafety cabinet for 1 h at room temperature (21°C) and approximately 10% relative humidity. Eighteen milliliters of 0.02% chlorine solution were poured onto the inoculated surface carriers and left for 1, 5, or 10 min following a previously developed protocol using the same surface carriers; this volume fully covers the surface (Gallandat et al. 2017). Each surface carrier was then placed in a prepared WhirlPak bag (Nasco, Fort Atkinson, Wisconsin) containing 300 mL of phosphate buffered saline (PBS, ) with 202 mg of sodium thiosulfate to neutralize chlorine. Any remaining disinfectant solution in the Petri dish was also poured into the WhirlPak bag, which is a modification of our previously published protocol to enhance bacteria recovery (Gallandat et al. 2017). All disinfection tests were carried out in triplicate. For each surface type, there was one negative control without test organism and three positive controls for which chlorine was replaced with 18 mL of PBS with sodium thiosulfate. If negative controls were not zero, then all data from that test run were discarded and the test was repeated. WhirlPak bags were kept on ice for no more than 5 h and processed as described in the section “E. coli and V. cholerae Sample Processing and Data Analysis.”
E. coli and V. cholerae Sample Processing and Data Analysis
Membrane filtration was performed using appropriate dilutions to detect culturable E. coli within 5 h of testing completion, using mColiBlue media (Hach, Loveland, Colorado) and 0.45 μm mixed cellulose membrane filters (Millipore Sigma, Germany). Petri dishes with pads were filled with 2 mL of media, then filters placed on top of the pads and incubated at 35°C for 24 h before counting colonies.
One hundred milliliters of each V. cholerae sample were processed within 5 h of testing through membrane filtration using 0.22 μm polycarbonate filters (Millipore Sigma, Germany), which were then placed in a 50-mL conical tube containing 12 mL of PBS and vortexed for 5 min to detach V. cholerae cells (Huq et al. 2012). Appropriate dilutions of the vortexed PBS were spread on thiosulfate-citrate-bile salts (TCBS) agar (BD Difco, Franklin Lakes, New Jersey) for selective detection of culturable V. cholerae and incubated at 35°C for 24 h before counting colonies.
Plate counts were analyzed in Microsoft Excel 2016 (Redmond, Washington State), including calculation of mean concentrations, remaining cfu per unit area, and standard errors. In case of nondetection, zero counts were replaced with half the theoretical detection limit, which was E. coli per carrier and V. cholerae, depending on surface carrier shape. If plates were “too numerous to count” (TNTC), a value of 250 cfu was assigned. LRVs calculated based on the minimum detection limit are reported as > calculated LRV and those calculated based on the maximum detection limit are reported as < calculated LRV. Additionally, recovery rates were calculated by comparing the difference between the inoculation and the positive control values for each surface.
Results and Discussion
Surface carriers were inoculated with E. coli and V. cholerae. Recovery rates after drying were 10%–20% from wood and 40%–100% from other surfaces. Positive control levels were thus E. coli and V. cholerae.
Exposure to 0.02% chlorine resulted in 1.3 to in culturable E. coli after 1, 5, and 10 min on all surfaces, and 4.9–7.0 LRV in culturable V. cholerae (Fig. 1; Table 1). E. coli LRV was higher at 10 min than 1 min on all surfaces except wood (which had the lowest recovery rate). This stratification of E. coli results supports pretesting results and the selected chlorine concentration. V. cholerae LRVs showed limited effect of exposure time and surface type. For culturable V. cholerae, LRVs were after 1 min and remained high across all surfaces with increasing exposure times. This indicates V. cholerae was inactivated more rapidly than E. coli on both nonporous and porous surfaces.
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| Surface type | Culturable E. coli (standard error) | Culturable V. cholerae (standard error) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Log (+) controls | 1 min LRV | 5 min LRV | 10 min LRV | Log (+) controls | 1 min LRV | 5 min LRV | 10 min LRV | |||||
| Nonporous | Heavy-duty tarp | 6.4 | (0.34) | 2.9 (0.54) | 4.7 (0.61) | 5.4 | (0.65) | 7.2 | (0.13) | 6.2 (0.90) | 6.2 (0.90) | 6.7 (0.34) |
| Nitrile | 6.7 | (0.25) | 3.5 (0.91) | 6.4 (1.45) | 6.7 | (0.14) | 7.1 | (0.17) | 6.8 (0.22) | 6.8 (0.22) | 7.0 (0.10) | |
| Stainless steel | 6.9 | (0.13) | 1.9 (0.08) | 3.5 (0.31) | 3.5 | (0.44) | 5.8 | (0.43) | 5.7 (0.25) | 5.2 (0.52) | 5.7 (0.25) | |
| Ceramic | 6.9 | (0.13) | 3.9 (0.50) | 6.9 (0.08) | 6.9 | (0.08) | 6.2 | (0.09) | 6.2 (0.05) | 6.2 (0.05) | 6.2 (0.05) | |
| Porous | Wood | 7.1 | (0.30) | 1.3 (0.24) | 3.3 (1.02) | 1.4 | (0.18) | 5.8 | (0.47) | 4.9 (0.51) | 4.9 (0.51) | 5.4 (0.27) |
| Foam | 7.0 | (0.36) | 1.4 (0.26) | 2.0 (0.41) | 3.7 | (1.01) | 7.1 | (0.14) | 6.5 (0.13) | 6.7 (0.08) | 6.3 (0.37) | |
We hypothesized that E. coli might be a surrogate for V. cholerae in surface disinfection efficacy testing. We found that V. cholerae is more sensitive to chlorine disinfection than culturable E. coli in almost all test conditions, excepting 5 and 10 min ceramic tests where no culturable organism was present for either test condition. However, the E. coli LRV was higher than V. cholerae because the inoculation concentration of E. coli was higher and is an artifact of testing. Our results thus support the use of culturable E. coli as a conservative surrogate for culturable V. cholerae in surface disinfection testing.
Existing literature provides context for these results. E. coli and V. cholerae both undergo similar inactivation mechanisms in response to hypochlorous acid (HOCl) involving oxidation of the elongation factor EF-TU (Wholey and Jakob 2012; Winter et al. 2008). EF-TU in V. cholerae is more sensitive to oxidative stress than E. coli EF-TU, suggesting EF-TU may increase chlorine susceptibility in V. cholerae (Wholey and Jakob 2012). This aligns with our results, and those of previous work, that culturable V. cholerae undergoes greater log reduction than culturable E. coli after water chlorination, and has a lower contact time and disinfectant concentration factor (CT-factor) (Adebisi et al. 2017; CDC 2019; Jones et al. 1992).
This work is limited in the following ways. Only one organism was evaluated for potential surrogacy. Of the limited existing research on surrogates for V. cholerae, two studies demonstrated promising findings with E. coli in suspension testing, thus we chose to focus on E. coli to expand that work to surface disinfection (Adebisi et al. 2017; Jones et al. 1992). Following WHO recommendations for surface disinfection in outbreak settings, we tested with a chlorine compound (NaDCC), which prior work has shown to be equivalent to other chlorine compounds in disinfection efficacy of E. coli and V. cholerae (Gallandat et al. 2017; String et al. 2020). Although we feel this was an appropriate choice to represent outbreak disinfectants, future work should expand on our testing with nonchlorine disinfectants commonly used in other settings. The concentration of chlorine solution tested was lower than WHO recommendations for surface disinfection; however, this was to ensure that enough E. coli cells survived for 1–10 min to evaluate surrogacy potential and should not affect the generalizability of our findings to higher concentrations given similar inactivation mechanisms in E. coli and V. cholerae. Finally, different detection limits and recoveries between test organisms and surfaces may have impaired comparisons.
As with several other pathogenic bacteria, V. cholerae can enter a viable but nonculturable (VBNC) state under stress in which it remains infectious but cannot grow on culture media (Chaiyanan et al. 2001). The role of VBNC V. cholerae in disease transmission is the subject of ongoing debate. VBNC V. cholerae has been isolated from surface water and it has been hypothesized that the VBNC stage is one mechanism through which V. cholerae is able to persist in the environment between outbreaks (Binsztein et al. 2004; Halpern et al. 2007; Lutz et al. 2013). There is evidence that VBNC V. cholerae can resuscitate, although laboratory studies in Bangladesh found culturable cells were the major contributor to waterborne transmission (Asakura et al. 2007; Mishra et al. 2018; Nelson et al. 2009). A laboratory assessment of V. cholerae’s persistence on surfaces found it was unculturable after 1–4 h but remained detectable in a VBNC state after 7 days on multiple nonporous and porous surfaces (Farhana et al. 2016). Overall, it is plausible that VBNC V. cholerae could play a role in disease transmission, including via fomites. Thus, epidemiological studies are recommended to quantify the role of surfaces in cholera transmission and to understand transmissibility and infectivity of VBNC V. cholerae.
Absent of evidence of any significant contribution by VBNC V. cholerae to epidemic transmission, based on the results herein, we propose the use of E. coli as a surrogate for culturable V. cholerae, which is a valuable contribution to disinfection research. Because bacteria in the VBNC state can withstand greater oxidative stress than corresponding culturable cells (Chaiyanan et al. 2001), our results do not support using E. coli as a surrogate for VBNC V. cholerae.
Conclusions
Overall, our results support the use of culturable E. coli as a conservative surrogate for culturable V. cholerae in surface disinfection testing. These results are particularly applicable to organizations and agencies implementing surface disinfection in health care facilities and households with cholera patients that have the ability to test for culturable E. coli using field methods (CDC 2010), but do not have the ability to test for V. cholerae using field methods.
Data Availability Statement
All data generated during the study appears in the published paper.
Acknowledgments
We would like to thank Dr. Amy Kahler, from the Centers for Disease Control and Prevention (Atlanta, Georgia), for providing initial training on methods used in this work. M. Zahrah’s time was covered by a Cataldo Fellowship from the Department of Civil and Environmental Engineering, Tufts University. Research and laboratory expenses were covered by a grant from ELRHA/R2HC entitled “Filling the Gap: Researching Commonly Implemented but Severely Under-Researched Water and Hygiene Interventions to Prevent Cholera Transmission.”
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Journal of Environmental Engineering
Volume 147 • Issue 4 • April 2021
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This work is made available under the terms of the Creative Commons Attribution 4.0 International license, https://creativecommons.org/licenses/by/4.0/.
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Received: May 9, 2020
Accepted: Nov 30, 2020
Published online: Jan 22, 2021
Published in print: Apr 1, 2021
Discussion open until: Jun 22, 2021
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