Tackling AMR in Wastewater Systems with Sneaky Bacteria

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Domestic wastewater treatment is among the main reasons why community health has improved dramatically since Victorian Times. Waste treatment plants (WTPs) effectively remove pathogens, carbon, and nitrogen, creating a healthier environment and reducing the waterborne infectious disease. However, WTPs were never designed to remove contemporary contaminants, such as antimicrobial resistant bacteria (ARB) or genes (ARG). Current WTPs reduce many ARBs/ARGs from wastes, but the "worst" sub-fraction of ARGs increase in WTPs, especially multi-ARGs (MRGs) that create the potential for indestructible pathogens. Researchers have been studying why multidrug resistance (MDR) is selected in WTPs. However, the cause is unknown, which impacts the long-term resilience of our water infrastructure.In the 1950s, German researchers observed a strange bacterial form in activated sludge (AS) in WTPs, called L-form bacteria. L-forms are "normal" bacteria that temporarily lose their cell wall. Although interesting, this observation was not pursued further. However, medical researchers recently discovered that L-form bacteria are common in MDR urinary tract (u-tract) infections, and my speculation is that L-form bacteria, which are intrinsically MDR, might be the "unknown" cause of MDR in WTP effluents. To test this bright idea, ~40 samples were collected from two UK WTPs and very high levels of L-form strains were found, especially in AS floc. Further, all L-form strains were putative "gut" bacteria, implying MDR in WTP effluents may be due to the selective survival of gut-originated L-form bacteria that "hide" in floc (in pseudo-dormant state) and then "sneak" back into WTP effluents because they survive waste treatment in their L-form state. The project do the following: - Develop better methods for detecting L-forms in wastewater;- Quantify environmental conditions in WTPs where L-form bacteria are selected and hide, and determine what triggers their reactivation;- Identify gene expression targets that promote/repress the L-form state and identify specific locations in WTPs where L-forms can be selectively destroyed; and- Perform bench- and pilot-scale reactor work to develop new treatment strategies to reduce MDR, especially aimed at reducing L-form survival in WTPs effluents.We already have >700 microbial MDR isolates from WTPs in the UK, Spain and India, although few have been tested for L-form development. However, early data suggest L-forms are common in AS, consistent with German observations. Within this context, work initially will focus on characterising our current MDR isolates in detail, especially categorising strains prone to L-formation and also identifying the presence and absence of key "L-form trigger genes" (in our isolates). Target genes will be refined and tested for diagnostics of L-forms, and also how their prevalence and local environments relate to MDR indictors (using qPCR, NGS and resistomics). With these data, structured sampling will be performed with three industrial partners on eight full-scale WTPs with different biological treatment technologies to identify "hot spots" of L-form selection and survival. Locale data will be used to guide lab- and pilot-scale testing of new and retrofit technologies to reduce MDR levels in WTP effluents, which will inform strategies for increasing resilience in our urban water infrastructure, especially reducing AMR spread and protecting community health. This proposal will deliver key outcomes for A Healthy and Resilient Nation, specifically H2, H3, R2 and R3, because it will generate basic and practical data that impacts all WTP designs in future; designs to reduce MRGs released to the environment. Beyond this outcome, transcendent discoveries will be made on the genetics, ecology and selection of L-form strains, which will inform the medical community on MDR infections and also improve diagnostics in both clinical and environmental settings.