Tackling AMR in Wastewater Systems with Sneaky Bacteria

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.

This project will transform approaches for reducing antimicrobial resistance (AMR), especially multidrug resistance (MDR) released from wastewater treatment plants (WTPs). The work will deliver outcomes contributing to a "Healthy" and "Resilient" Nation, specifically addressing H2 (improve prevention), H3 (optimise diagnosis), R2 (ensure a reliable infrastructure), and R3 (develop better solutions to health threats). It will use a wholly new idea from cell biology (i.e., selection and survival of L-from bacteria) to develop new and less costly engineering options for reducing MDR in WTPs, which could change the way we view AMR and wastewater treatment in future applications.

Although this project will immediately benefit the water industry, it also benefits other sectors, including city, national and international governments; the public health sector; regulatory agencies; the pharmaceutical industry; and society as a whole. Specifically, growing concern exists over increasing AMR in human and veterinary medicine, and mounting evidence suggests inadequately treated wastewater significantly impacts AMR in the environment, even in the UK. We deservedly trust our WTPs, but they still release disproportionately high levels of MDR, which this project aims to address from a wholly new perspective.

Each sector will benefit in different ways. The public health sector will be provided new knowledge on root causes of MDR in WTPs, and also will be provided new biochemical and genetic data needed to develop better diagnostic tools for MDR detection in clinical and environmental samples. The pharmaceutical industry will benefit by our solutions reducing the spread of AMR through more resilient WTPs, making drug discovery more justifiable and extending the commercial-life of existing antimicrobials. Regulatory agencies (e.g., local governments, environment agencies) will gain by being provided better informed, scientifically robust metadata essential to develop environmental risk management guidance related to AMR and WTPs. As a result, society as a whole will benefit by improved resilience in current and future WTP designs, specifically reducing AMR released in effluents to our water environment.

A wide range of vehicles will be employed to maximise impact of the project. The project includes water industry stakeholders, which will act as an ad hoc Project Advisory Group, providing advice, guidance, access to WTPs, and a pathway for industrial outreach on the project. Impact also will be promoted by publication in leading environmental publications, and presentation at environmental AMR and microbiology meetings, including EDAR5 in Hong Kong and ISME17 in Leipzig. Further, our existing network of contacts will be used disseminate findings to national and international organisations, including the WHO and PACCARB in the US with whom the PI already works. Finally, social and traditional media (TV and radio) will be employed to instantly provide information of community value to the wider public; platforms on which our group has been very successful in the recent past.

Overall, this project with reconsider what fundamentally drives MDR in bacteria, particularly altering their form to an L-form state, which renders bacteria in WTP effluents intrinsically MDR. Through such discoveries and wide stakeholder connections, impact and dissemination of work will be assured, including translation to other disciplines, such as MDR diagnosis and prevention in medicine. However, the ultimate aim is to use engineering to create a healthier, resilient environment in the UK, but then to extend those benefits, which have significant altruistic and economic value, to the wider world.