Download PDF Winter Bridge on The Grainger Foundation Frontiers of Engineering December 13, 2024 Volume 54 Issue 4 This issue features articles by The Grainger Foundation US Frontiers of Engineering 2024 symposium participants. The articles examine cutting-edge developments in microbiology and health, artificial intelligence, the gut-brain connection, and digital twins. Designing Waste Management Systems to Prevent the Spread of Antibiotic Resistance: Challenges and Opportunities Friday, December 13, 2024 Author: Ishi Keenum This article reviews the current state of the science on engineering interventions to reduce antibiotic resistance and highlights measurement challenges and opportunities for antibiotic resistance. Antibiotic resistance (AR) is a global health threat that challenges the efficacy of many critical medications. Traditionally, AR has been primarily considered a clinical challenge. However, a scientific consensus has arisen around the role that industry, society, and agriculture also play in the dissemination of AR. In response to this, a One Health approach has been adopted by the United Nations that highlights the interconnectedness of humans, agriculture, and the environment (figure 1). This framework prioritizes the mitigation of AR sources into the environment while continuing to reduce antibiotic prescriptions when efficacious. The goal of this article is to present the current state of the science on engineering interventions to reduce AR from anthropogenic sources and highlight the measurement challenges and opportunities present to effectively implement AR monitoring and mitigation. The Environmental Dimension of AR Surface waters are critical aquatic environments that receive sources of AR from wastewater treatment, industry, recreational activities (Leonard et al. 2018; Nappier et al. 2020), and agricultural runoff. Currently, no regulations exist globally that specifically target antibiotic-resistance genes (ARGs) or antibiotic-resistant bacteria (ARB) to surface waters. Numerous studies have shown that environments receiving municipally and agriculturally derived wastewaters contain more ARGs and ARB than unimpacted environments (Lee et al. 2021; Pruden et al. 2006; Rizzo et al. 2013). Specific genes such as sul1, a sulfonamide resistance gene, and intI1, a mobile genetic element that encodes for the class I integron gene, have been identified as key indicators of anthropogenic activity (Gillings et al. 2015; Pei et al. 2006), while tetracycline resistance genes have been identified in surface waters as indicative of agricultural runoff (Schmitt et al. 2006). Land use has also been identified as a key factor in environmental AR (Keely et al. 2022). Recent work has shown that clinical isolates contributing to a hospital outbreak can be cultured downstream of wastewater utilities, highlighting the potential persistence and viability of clinically relevant organisms in surface waters (Loudermilk et al. 2022). It is widely accepted that inadequately treated wastewaters and fecal sources (including leaky septic tanks, non-point source agriculture pollution, and combined sewer overflows) serve as critical contribution points of AR to surface waters, leading to the need for interventions on how to best engineer wastewater systems to reduce AR. Engineering Wastewater Treatments to Mitigate AR Wastewater treatment plants (WWTPs) have been identified as key convergence points of AR due to the contribution of municipal, hospital, and industrial waste. Further, WWTPs have been suggested to be key evolutionary points where ARGs can come into contact with human pathogens as well as aquatic bacteria (Brown et al. 2024). While many studies have examined the potential of ARG and ARB treatment removal (Rizzo et al. 2013), there is no consensus on what should be best measured for AR and what represents an appropriate reduction or “safe” discharge limit. This is critical as not all ARGs or ARB behave similarly, and some ARGs are so widespread that they are no longer clinically relevant. Despite these challenges, most trends for ARB and ARG removal are similar to other conventional microbial indicators such as E. coli. However, recent data on disinfection processes and advanced oxidation processes demonstrate that these processes, specifically, may enrich for ARB and ARGs (Ashbolt et al. 2018). Further, while removal of ARB and ARGs may be measured at treatment effluent outfall, bacteria may be able to regrow when the treatment pressure is removed. Recent work by Keenum and colleagues measured the removal of ARGs and ARB in two full-scale water reuse systems and found that, in the non-potable water reuse system, significant regrowth occurred in the distribution system across all measurement methods (Keenum et al. 2024). This highlights how, as we consider new ways to improve sustainability and water usage, we need to be particularly mindful of emerging contaminants. Developing a Risk Assessment Framework for AR Chemical and microbiological contaminants are assessed for safe levels through a process called risk assessment. Specifically, quantitative microbial risk assessment (QMRA) is used to estimate the risk of infection and illness when a population is exposed to microorganisms in the environment. A challenge in developing QMRA models for AR is that each model is developed for a target organism, and these are built on experimentally derived data. A common challenge across QMRA studies is obtaining enough data to confidently assess a specific scenario. Recent work by Schoen and colleagues assessed the risk of Methicillin-resistant Staphylococcus aureus colonization and disease burden from potable and non-potable water reuse scenarios and found it was below the health benchmark of 10-4 infections per year. However, they acknowledge that this may not be the case in conventional WWTP effluent or in greywater. Regardless, the authors acknowledge the need for further studies to investigate the potential risk of AR. Most QMRA frameworks are based on viable culturable organisms. However, much of the ARG data is molecular, and researchers measured the occurrence of a gene rather than the occurrence of an organism. Further work is needed to integrate ARGs into QMRA models. Measurement Challenges in AR When evaluating AR, it is critical to recognize that different measurement methods inform different aspects of ARB or ARGs within a system. Culture-based methods can provide essential information regarding survival and growth of ARB and are directly informative for QMRA models. However, culture-based methods are only effective for microbes that can be cultured in laboratory settings, which represent a small fraction of environmental bacteria. Culture methods are incredibly valuable as they enable the identification of newly evolved ARB and ARGs because they select based on phenotypic presence of AR as opposed to predetermined genetic indicators (Chaudhry et al. 2017; Zhiteneva et al. 2020). A major advantage of culture-based methods is the inherent confirmation that the target is viable and the opportunity to conduct further testing, such as multi-drug resistance testing or whole genome sequencing analysis, as a means to track specific sources and identify carriage of specific ARGs and where and how these were acquired (Hegreness et al. 2008). As we consider new ways to improve sustainability and water usage, we need to be particularly mindful of emerging contaminants. In contrast to culture-based methods, molecular methods enable the detection of ARGs across all DNA in all bacteria present, which is especially important in complex environmental systems. Molecular methods, including quantitative polymerase chain reaction (qPCR, inclusive of newer digital PCR methods) and shotgun metagenomics, can complement culture methods by comprehensively profiling all ARGs in a sample. qPCR methods can be used to directly quantify the removal, selection pressures (e.g., presence of antibiotics or other stressors), and/or horizontal gene transfer that may elevate or reduce the absolute abundances of genes and variants at extremely low detection rates (down to one gene copy per microliter of DNA). A key challenge in utilizing PCR for the quantification of ARGs is that genes must be pre-identified and there are no standardized monitoring targets (Rocha et al. 2019). Next-generation sequencing has emerged as an incredibly powerful methodology that enables untargeted detection of AR. While, previously, sequencing was considered a semi-quantitative methodology, recently, quantitative metagenomic sequencing (qMeta) has emerged as an untargeted way to simultaneously quantify all genes present in a sample. Progress Made in Standardization of AMR Monitoring Researchers and the international community are working to identify common monitoring targets to develop databases of effective AR mitigation strategies. The Advisory Group for Integrated Surveillance of Antimicrobial Resistance of the World Health Organization has proposed testing extended spectrum beta-lactamase (i.e., cefotaxime)-resistant E. coli as an internationally standardized monitoring target for water and wastewater towards a larger goal of integrated One Health antimicrobial resistance surveillance (Matheu et al. 2017). Extended spectrum beta-lactamase E. coli monitoring has been adopted by the 27 countries forming the Joint Programming Initiative on Antimicrobial Resistance for water and sanitation monitoring purposes. Additionally, work has been done to identify key ARGs that can be monitored by qPCR methods in specific scenarios (Keenum et al. 2021; Liguori et al. 2023). While clinical intervention and novel antibiotic development are critical, it is equally critical to ensure that environmental sources of antibiotic resistance are mitigated, else new drugs will quickly become obsolete. Conclusions AR is a growing global challenge that is anticipated to cause 8.22 million deaths per year by 2050 (Naghavi et al. 2024). While clinical intervention and novel antibiotic development are critical, it is equally critical to ensure that environmental sources of AR are mitigated, else new drugs will quickly become obsolete. Surface waters are key points of recreation and potential exposure to ineffectively treated effluents. WWTPs have been identified as key convergence and evolution points for AR. However, challenges remain in developing QMRA models for ARGs as well as establishing monitoring criteria in the environment. References Ashbolt N, Pruden A, Miller J, Riquelme MV, Maile-Moskowitz A. 2018. Antimicrobial resistance: Fecal sanitation strategies for combatting a global public health threat. In: Water and Sanitation for the 21st Century: Health and Microbiological Aspects of Excreta and Wastewater Management (Global Water Pathogens Project). Rose JB, Jiménez-Cisneros B, eds. (Part 3: Specific Excreted Pathogens: Environmental and Epidemiology Aspects - Section 2: Bacteria. Pruden A, Ashbolt N, Miller J, eds). UNESCO, Michigan State University. Brown CL, Maile-Moskowitz A, Lopatkin AJ, Xia K, Logan LK, Davis BC, Zhang L, Vikesland PJ, Pruden A. 2024. Selection and horizontal gene transfer underlie microdiversity-level heterogeneity in resistance gene fate during wastewater treatment. Nature Communications 15(1):5412. Chaudhry RM, Hamilton KA, Haas CN, Nelson KL. 2017. Drivers of microbial risk for direct potable reuse and de facto reuse treatment schemes: The impacts of source water quality and blending. International Journal of Environmental Research and Public Health 14(6):1–20. Gillings MR, Gaze WH, Pruden A, Smalla K, Tiedje JM, and Zhu Y-G. 2015. Using the Class 1 integron-integrase gene as a proxy for anthropogenic pollution. The ISME Journal 9(6):1269. Hegreness M, Shoresh N, Damian D, Hartl D, and Kishony R. 2008. Accelerated Evolution of Resistance in Multidrug Environments. Proceedings of the National Academy of Sciences 105(37):13977–81. Keely SP, Brinkman NE, Wheaton EA, Jahne MA, Siefring SD, Varma M, Hill RA, Leibowitz SG, Martin RW, Garland JL, Haugland RA. 2022. Geospatial patterns of antimicrobial resistance genes in the US EPA national rivers and streams assessment survey. Environmental Science & Technology 56(21):14960–14971. Keenum I, Calarco J, Majeed H, Hager E, Bott C, Garner E, Harwood VJ, Pruden A. 2024. To what extent do water reuse treatments reduce antibiotic resistance indicators? A comparison of two full-scale systems. Water Research 254(1):121425. Keenum I, Liguori K, Calarco J, Davis BC, Milligan E, Harwood VJ, Pruden A. 2021. A framework for standardized qPCR-targets and protocols for quantifying antibiotic resistance in surface water, recycled water and wastewater. Critical Reviews in Environmental Science and Technology 52(24):4395–4419. Lee J, Ju F, Maile-Moskowitz A, Beck K, Maccagnan A, McArdell CS, Molin MD, Fenicia F, Vikesland PJ, Pruden A, and 2 others. 2021. Unraveling the riverine antibiotic resistome: The downstream fate of anthropogenic inputs. Water Research 197:117050. Leonard AFC, Zhang L, Balfour AJ, Garside R, Hawkey PM, Murray AK, Ukoumunne OC, Gaze WH. 2018. Exposure to and colonisation by antibiotic-resistant E. coli in UK coastal water users: Environmental surveillance, exposure assessment, and epidemiological study (Beach Bum Survey). Environment International 114:326–33. Liguori K, Keenum I, Davis B, Milligan E, Heath LS, Pruden A, Calarco J, Harwood VJ. 2023. Standardizing Methods with QA/QC Standards for Investigating the Occurrence and Removal of Antibiotic Resistant Bacteria/Antibiotic Resistance Genes (ARB/ARGs) in Surface Water, Wastewater, and Recycled Water. Water Research Foundation. Loudermilk EM, Kotay SM, Barry KE, Parikh HI, Colosi LM, Mathers AJ. 2022. Tracking Klebsiella pneumoniae carbapenemase gene as an indicator of antimicrobial resistance dissemination from a hospital to surface water via a municipal wastewater treatment plant. Water Research 213:118151. Matheu J, Aidara-Kane A, Andremont A. 2017. The ESBL Tricycle AMR Surveillance Project: A Simple, One Health Approach to Global Surveillance. World Health Organization (WHO) Advisory Group for Integrated Surveillance of Antimicrobial Resistance (AGISAR). Naghavi M, Vollset SE, Ikuta KS, Swetschinski LR, Gray AP, Wool EE, Aguilar GR, Mestrovic T, Smith G, Han C et al. 2024. Global burden of bacterial antimicrobial resistance 1990–2021: A systematic analysis with forecasts to 2050. The Lancet 404(10459):1199–1226. Nappier SP, Liguori K, Ichida AM, Stewart JR, Jones KR. 2020. Antibiotic resistance in recreational waters: State of the science. International Journal of Environmental Research and Public Health 17(21):8034. Pei R, Kim S-C, Carlson KH, Pruden A. 2006. Effect of river landscape on the sediment concentrations of antibiotics and corresponding antibiotic resistance genes (ARG). Water Research 40(12):2427–35. Pruden A, Pei R, Storteboom H, Carlson KH. 2006. Antibiotic resistance genes as emerging contaminants: Studies in northern Colorado. Environmental Science & Technology 40(23):7445–50. Rizzo L, Manaia C, Merlin C, Schwartz T, Dagot C, Ploy MC, Michael I, Fatta-Kassinos D. 2013. Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: A review. Science of the Total Environment 447:345–60. Rocha J, Fernandes T, Riquelme MV, Zhu N, Pruden A, Manaia CM. 2019. Comparison of culture-and quantitative PCR-based indicators of antibiotic resistance in wastewater, recycled water, and tap water. International Journal of Environmental Research and Public Health 16 (21):4217. Schmitt H, Stoob K, Hamscher G, Smit E, Seinen W. 2006. Tetracyclines and tetracycline resistance in agricultural soils: Microcosm and field studies. Microbial Ecology 51 (3):267–76. United Nations Environment Programme. 2023. Bracing for Superbugs: Strengthening Environmental Action in the One Health Response to Antimicrobial Resistance. United Nations. Zhiteneva V, Hübner U, Medema GJ, Drewes JE. 2020. Trends in conducting quantitative microbial risk assessments for water reuse systems: A review. Microbial Risk Analysis 16:100132. About the Author:Ishi Keenum is assistant professor in the Civil, Environmental, and Geospatial Engineering Department at Michigan Technological University.