Impact of stereochemistry of antimicrobial agents on their environmental fate, biological potency and the emergence of resistance

Lead Research Organisation: University of Bath
Department Name: Chemistry

Abstract

This project aims to understand and address the impact of stereoisomerism of antimicrobial agents in their environmental cycle on mechanisms behind the development of antimicrobial resistance. The risk of promotion of antibiotic resistant bacteria is by far the greatest human health concern with regards to medicinal products in the environment. The continuous introduction of sub-inhibitory quantities of antimicrobial agents (AAs) to the environment is believed to be directly linked with antimicrobial resistance (AMR). Unfortunately, there is little knowledge of mechanisms in the environment and influencing factors due to the multi-dimensional nature of the AMR problem. There are several research gaps that need to be addressed including research into contaminated habitats (e.g. wastewater) where AAs, co-selecting agents, bacteria carrying resistance determinants and favourable conditions for bacterial growth prevail at the same time. Furthermore, the stereochemistry of AAs (which is key in defining their biological potency) has never been studied in the context of their environmental fate and effects. This is an oversight as changes in stereoisomeric profile of AAs throughout their environmental cycle will lead to (and be influenced by) changes in the composition and structure of microbial communities present in the environment. This might further contribute to the development of AMR, a phenomenon that has never been the subject of investigation in the context of stereochemistry of AAs.

This project postulates that stereochemistry of AAs determines their environmental fate and biological effects. It also hypothesizes that two enantiomers of the same AA should be recognised as two different substances that can elicit different responses leading to changes in the environmental fate and effects of the drug.

The project will:
1. Verify the mechanisms of (stereoselective) transformation of chiral antimicrobial agents and their metabolites during wastewater treatment and in receiving waters
2. Identify resistant bacterial taxa responsible for (stereoselective) degradation of antimicrobial agents and to study the development of antimicrobial resistance at stereoisomeric level
3. Recommend changes to ERA via inclusion of AAs (and their stereochemistry) and ARGs as AMR indicators

The stereochemistry of AAs is complex, as many of the semi-synthetic agents are marketed as mixtures of diastereomers and a number of synthetic agents are used as racemates. In this project we will focus on ofloxacin and chloramphenicol, but we will also consider other synthetic quinolones, Beta-lactams (e.g. amoxicillin) and carbapenems (e.g. meropenem).

Considering the importance of better understanding environmental and human health impacts from chiral pollutants such as AAs and the need for the development of new solutions tackling AMR, this project has the potential to lead to groundbreaking research with long term scientific, technological and societal impact.

Planned Impact

This project will provide cutting-edge advances in the understanding of the transformation mechanisms of antimicrobial agents and their biological effects including AMR. It will identify future research objectives directed at informed decision making on the wastewater treatment technologies used, the procedures applied in risk assessment of antimicrobial agents, the use of antimicrobial agents, relevant policies/regulations and consumer behaviours.

This project partners with a major UK water utility - Wessex Water, which will provide the opportunity for the project outcomes to be translated into effective applications within the water industry such as revision and implementation of existing and novel strategies aiming at reduction of impacts from antibiotics and AMR. In the light of the research, there may be grounds for legislative, procedural or policy changes in the area of wastewater treatment which are likely to be of significant interest to regulators. Collaboration with Environment Agency will allow for the development of a strategy aiming at limiting impacts from antibiotics and AMR.

Impact activities will include:
1. Development of inter-disciplinary and cross-sectoral research capability in AMR at the University of Bath.
2. Enhancing public-private sector collaborations: this project builds upon existing collaborations between public organisations and the private sector and will enhance these collaborations through the active involvement of the private sector in research activities. A workshop will be organised to facilitate knowledge exchange in particular in areas of pharmaceutical research, environmental management and water engineering.
3. Strengthening interdisciplinary and cross-sectoral collaborations in research: Due to its interdisciplinary nature, this project requires cross-sectoral collaborations in research both in formulating project objectives and in implementing research outcomes with an aim of measurable short and long-term impact in several AMR related areas including: water engineering, regulatory, pharmaceutical and health related sectors. This will be achieved via inclusion of key stakeholders as Project Partners representing water (Wessex Water) and regulatory (Environment Agency) sectors.
4. Communication of research outputs via publications in high IF journals, presentations at scientific conferences, popular media, articles in newspapers, webpages, presentations to the general public (e.g. Bath Science Café or Pint of Science talks), workshops and collaborative visits (to facilitate knowledge exchange between academic and public/private sector) and other outreach activities (e.g. promotion of research at schools).
5. Acquisition of key skills by the project researchers through a unique training programme that involves engagement with integrated disciplines conducting cutting edge research and exposure to private sector.

Publications

10 25 50
 
Description Output 1:
A new analytical method has been developed to undertake the multiresidue enantiomeric profiling of (fluoro)quinolones and their metabolites in solid and liquid environmental matrices using chiral HPLC-MS/MS method and a CHIRALCEL ® OZ-RH column. Simultaneous chiral separation was obtained for chiral ofloxacin and its main
metabolites ofloxacin-N-oxide and desmethyl-ofloxacin; moxifloxacin; the prodrug prulifloxacin and its active compound ulifloxacin; flumequine; nadifloxacin and R-(þ)-besifloxacin. Achiral antibiotics (ciprofloxacin, norfloxacin and nalidixic acid) were also included in the method to enable the analysis of all targeted quinolones within one analytical run. Satisfactory enantiomeric resolution was obtained for five out of eight chiral drugs enabling quantitative analysis. The overall performance of the
method was satisfactory with a method precision <20%, relative recoveries >70% for most of the analytes and method detection limits (MDL) at low ng/L levels. Enantiomeric profiling from a week-long monitoring campaign in the UK showed that (±)-ofloxacin was found to be racemic in upstream waters but it was enriched with S-(-)-enantiomer in wastewater and in receiving waters. This could be due to the fact that ofloxacin can be used both as a racemate and as a S-(-)-enantiomer. Its consumption was further confirmed by the chiral signature of the investigated ofloxacin metabolites. As a result, alterations in the enantiomeric composition of antibiotics could influence not only their activity and toxicity in the environment, but also could induce changes in the microbial communities constantly exposed to them.

Output 2:
This study provided an insight into the prevalence of (fluoro)quinolones (FQs) and their specific quinolone qnrS resistance gene in the Avon river catchment area receiving treated wastewater from 5 wastewater treatment plants (WWTPs), serving 1.5 million people and accounting for 75% of inhabitants living in the catchment area in the South West of England. Ofloxacin, ciprofloxacin, nalidixic acid and norfloxacin were found to be ubiquitous with daily loads reaching a few hundred g/day in wastewater
influent and tens of g/day in receiving waters. This was in contrast to other FQs analysed: flumequine, nadifloxacin, lomefloxacin, ulifloxacin, prulifloxacin, besifloxacin and moxifloxacin, which were hardly quantified. Enantiomeric profiling revealed that ofloxacin was enriched with the S-(-)-enantiomer, likely deriving from its prescription as the more potent enantiomerically pure levofloxacin, alongside racemic ofloxacin. While ofloxacin's enantiomeric fraction (EF) remained constant, high stereoselectivity was
observed in the case of its metabolite ofloxacin-N-oxide. The removal efficiency of quinolones during wastewater treatment at 5 WWTPs utilising either trickling filters (TF) or activated sludge (AS), was compound and wastewater treatment process dependent, with AS providing better efficiency than TF. The qnrS resistance gene was ubiquitous in wastewater. Its removal was WWTP treatment process dependent with TF performing best and resulting in significant removal of the gene (from 28 to 75%). AS
underperformed with only 9% removal in the case of activated sludge and actual increase in the gene copy number within sequencing batch reactors (SBRs). Interestingly, the data suggests that higher removal of antibiotics could be linked with high prevalence of the gene (SBR and WWTP E) and vice versa, low removal of antibiotic is correlated with lower prevalence of the gene in wastewater effluent (TF, WWTP B and D). This is especially prominent in the case of ofloxacin and could indicate that AS might be facilitating antimicrobial resistance (AMR) prevalence to higher extent than TF. Wastewater based epidemiology (WBE) was also applied to monitor any potential misuse (e.g. direct disposal) of FQs in the catchment. In most cases higher use of antibiotics with respect to official statistics (i.e. ciprofloxacin, ofloxacin) was observed, which suggests that FQs management practice require further attention.
Exploitation Route Output 1: Analytical methods can be utilised by researchers and water/analytical/environment professionals, including regulators, to study fate and effects of chiral antibiotics.
Output 2: Knowledge of enantiomer-depended processes occurring in the environment will be of importance to those working in the area of environmental and public health risks, including regulators.
Sectors Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description This award has contributed to the following: • Improved Wessex Water's ability to detect and analyse a wider range of compounds and their metabolites using new rapid analytical techniques; improved sampling methods and influenced sampling sites; • Informed Wessex Water priorities including investment decisions and future areas of work i.e., the AMR programme (the choice of site for UKWIR AMR and methods); • Impact of the AMR programme to understand current sources, transformation through the sewerage system and potential risks to both wider public health and the environment. • Contributed to Wessex Water shift in focus from characterising wastewater to looking at the wider impact on the environment, ecosystem and public health • Raised awareness and understanding of the wider impact of contaminants in our sewage and receiving watercourses
First Year Of Impact 2019
Sector Environment
Impact Types Policy & public services

 
Description DTG Directed, Stereochemistry of antimicrobial agents in urban water cycle and its impacts upon the evolution of antimicrobial resistance
Amount £89,114 (GBP)
Funding ID NE/R008094/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 09/2018 
End 09/2022
 
Description EPSRC, Project title: The fate of antimicrobials in urban wastewater and its role in the development of antimicrobial resistance
Amount £23,500 (GBP)
Funding ID 1789298 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 09/2016 
End 12/2019
 
Title New analytical methodology for enantioselective separation of quinolones in environmental matrices 
Description New analytical methodology utilising chiral chromatography and mass spectrometry to separate chiral and non-chiral (fluoro)quinolones in environmental matrices 
Type Of Material Improvements to research infrastructure 
Year Produced 2018 
Provided To Others? Yes  
Impact This method allowed for understanding of enantioselective processes of (fluoro)quinolones in the environment 
URL https://www.sciencedirect.com/science/article/pii/S0045653518308476?via%3Dihub
 
Title SAM (stereoselective antimicrobial metabolism workflow 
Description SAM (stereoselective antimicrobial metabolism) workflow is a novel interdisciplinary approach for assessing bacterial resistance mechanisms in the context of antibiotic-bacteria interactions that utilise a combination of whole genome sequencing and mass spectrometry 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? Yes  
Impact n/a 
URL https://www.frontiersin.org/articles/10.3389/fmicb.2021.562157/full
 
Title Concentrations of antibiotic resistance genes in wastewater and receiving waters following water treatment in SW England, June-October, 2015 
Description Prevalence of quinolone qnrS resistance gene in the aquatic environment from the Avon river catchment area receiving treated wastewater from 5 wastewater treatment plants (WWTPs), serving 1.5 million people and accounting for 75% of inhabitants living in the catchment area in the South West of England. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact n/a 
URL https://catalogue.ceh.ac.uk/documents/102f8141-2a9a-4ffd-89f6-961af36ddcb3
 
Title Concentrations of antibiotics in wastewater and receiving waters following water treatment in SW England, June-October, 2015 
Description his dataset comprises of weekly concentrations of fluoroquinolones in wastewater and receiving waters collected in the Avon river catchment South West UK. The following fluoroquinolones were analysed: (±)-ofloxacin, (±)-ofloxacin-N-oxide, (±)-desmethyl-ofloxacin, (±)-lomefloxacin, (±)-moxifloxacin, S,S-moxifloxacin-N-sulfate, R-(+)-besifloxacin, (±)-prulifloxacin, (±)-ulifloxacin, (±)-flumequine and (±)-nadifloxacin; ciprofloxacin, desethylene-ciprofloxacin, norfloxacin and nalidixic acid. The concentrations of (fluoro)quinolones were measured in receiving treated wastewater from 5 wastewater treatment plants (WWTPs), which serves 1.5 million people accounting for 75% of inhabitants living in the catchment area in the South West of England. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact n/a 
URL https://catalogue.ceh.ac.uk/documents/f4c79bd3-3ce9-4f4e-ae19-bb4fd2dce7a5
 
Description Collaboration with Environment Agency 
Organisation Environment Agency
Country United Kingdom 
Sector Public 
PI Contribution Collaboration within NE/N019261/1 project entitled: 'Impact of stereochemistry of antimicrobial agents on their environmental fate, biological potency and the emergence of resistance'
Collaborator Contribution Collaboration within NE/N019261/1 project entitled: 'Impact of stereochemistry of antimicrobial agents on their environmental fate, biological potency and the emergence of resistance'
Impact Ongoing
Start Year 2016
 
Description Collaboration with Wessex Water 
Organisation Wessex Water
Country United Kingdom 
Sector Private 
PI Contribution expertise, equipment, facilities
Collaborator Contribution expertise, equipment, facilities
Impact Several, including academic papers: Petrie, B., Proctor, K., Youdan, J., Barden, R. and Kasprzyk-hordern, B., 2017. Critical evaluation of monitoring strategy for the multi-residue determination of 90 chiral and achiral micropollutants in effluent wastewater. Science of the Total Environment, 579, pp. 569-578. Petrie, B., Gravell, A., Mills, G. A., Youdan, J., Barden, R. and Kasprzyk-Hordern, B., 2016. In situ calibration of a new chemcatcher configuration for the determination of polar organic micropollutants in wastewater effluent. Environmental Science & Technology, 50 (17), pp. 9469-9478. Petrie, B., Youdan, J., Barden, R. and Kasprzyk-Hordern, B., 2016. A new framework to diagnose the direct disposal of prescribed drugs in wastewater - a case study of the antidepressant fluoxetine. Environmental Science & Technology, 50 (7), pp. 3781-3789. Petrie, B., Youdan, J., Barden, R. and Kasprzyk-Hordern, B., 2016. Multi-residue analysis of 90 emerging contaminants in liquid and solid environmental matrices by ultra-high-performance liquid chromatography tandem mass spectrometry. Journal of Chromatography A, 1431, pp. 64-78. Petrie, B., Barden, R. and Kasprzyk-Hordern, B., 2015. A review on emerging contaminants in wastewaters and the environment:current knowledge, understudied areas and recommendations for future monitoring. Water Research, 72, pp. 3-27.
Start Year 2012
 
Description What's Your water telling you? Urban water profiling inform the state of the environment and public health 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public talk delivered by Kasprzyk-Hordern B entitled: 'What's Your water telling you? Urban water profiling inform the state of the environment and public health', RSC Event, Bristol, 13th Feb 2018, University of the West of England
Year(s) Of Engagement Activity 2017
 
Description 3. Cartwright, N and Kasprzyk-Hordern, B. To understand the risks to the environment from chemicals, , Environment Agency meeting 'Working Together for a Better Environment', Bristol, 21st February 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Talk on collaboration between University of Bath, Wessex Water and Environment Agency
Year(s) Of Engagement Activity 2017
URL https://www.instituteofwater.org.uk/events-info/613/
 
Description Innovation through partnership working, DEFRA, London 15th November 2016. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Talk at DEFRA WQ Team away day
Year(s) Of Engagement Activity 2016
 
Description Kasprzyk-Hordern, Antibiotics and resistance genes in the environment, University of Nottingham Sutton Bonington AMR Webinar Series 2020 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Talk by Kasprzyk-Hordern, Antibiotics and resistance genes in the environment, University of Nottingham Sutton Bonington AMR Webinar Series 2020
Year(s) Of Engagement Activity 2020
 
Description Kasprzyk-Hordern, interview, BBC Radio Wales: Science Cafe, 8th of September 2020 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Radio interview
Year(s) Of Engagement Activity 2020
URL https://tinyurl.com/y52g4bld
 
Description Public talk entitled: 'Healthy waters: Pharmaceuticals in the environment - cause for concern? Bath Festival of Nature, 2nd June 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public talk delivered by Kasprzyk-Hordern. Title: Healthy waters: Pharmaceuticals in the environment - cause for concern? Bath Festival of Nature, 2nd June 2017.
Year(s) Of Engagement Activity 2017
 
Description Talk by B. Kasprzyk-Hordern, 'Urban water fingerprinting for environmental and public health assessmen', Kwaluzu-Natal University, Durban, 1st of February 2019, South Africa 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Talk by B. Kasprzyk-Hordern, 'Urban water fingerprinting for environmental and public health assessmen', Kwaluzu-Natal University, Durban, 1st of February 2019, South Africa (invited talk)
Year(s) Of Engagement Activity 2019
 
Description Talk by B. Kasprzyk-Hordern, My career in water, 18th UK Young Water, Professionals Conference, University of Bath, 10 -12th April 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Talk by B. Kasprzyk-Hordern, My career in water, 18th UK Young Water, Professionals Conference, University of Bath, 10 -12th April 2017
Year(s) Of Engagement Activity 2017
 
Description Talk by Kasprzyk-Hordern 'Antimicrobials in water cycle: Research gaps', Israel-UK Synergy programme grant symposium and workshop, 25th and 26th April 2018:"One Health: antimicrobial resistance from mechanisms to interdisciplinary solutions" The Institute of Microbiology and Infection, University of Birmingham 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Kasprzyk-Hordern, B., Castrignano E, Edler, F, 'Antimicrobials in water cycle: Research gaps', Israel-UK Synergy programme grant symposium and workshop, 25th and 26th April 2018:"One Health: antimicrobial resistance from mechanisms to interdisciplinary solutions" The Institute of Microbiology and Infection, University of Birmingham (invited talk)
Year(s) Of Engagement Activity 2018
 
Description Talk by Kasprzyk-Hordern, 'Urban water fingerprinting to inform the state of the environment and public health', University College London Chemical & Physical Society Guest Lecturer 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact B. Kasprzyk-Hordern, 'Urban water fingerprinting to inform the state of the environment and public health', University College London Chemical & Physical Society Guest Lecturer 16th October 2018 (invited talk)
Year(s) Of Engagement Activity 2018
 
Description Talk by Kasprzyk-Hordern, B., What's Your water telling you? Urban water profiling inform the state of the environment and public health, RSC Event, Bristol, 13th Feb 2018, University of the West of England 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Kasprzyk-Hordern, B., What's Your water telling you? Urban water profiling inform the state of the environment and public health, RSC Event, Bristol, 13th Feb 2018, University of the West of England (invited talk)
Year(s) Of Engagement Activity 2018
 
Description Talk by Kasprzyk-Hordern, Pharmaceuticals in the environment - cause for concern?, Viewpoints Keynsham: Healthy Water, 23rd of March 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public talk by Kasprzyk-Hordern, Pharmaceuticals in the environment - cause for concern?, Viewpoints Keynsham: Healthy Water, 23rd of March 2017
Year(s) Of Engagement Activity 2017
 
Description Talk delivered by Kasprzyk-Hordern, entitled: Water fingerprinting for public health assessment', Wetenschappelijke Advies Raad / Scientific Advisory Council KWR, Nieuwegein, the Netherlands, 1st of June 2017 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Talk delivered by Kasprzyk-Hordern, entitled: Water fingerprinting for public health assessment' at Wetenschappelijke Advies Raad / Scientific Advisory Council KWR, Nieuwegein, the Netherlands, 1st of June 2017
Year(s) Of Engagement Activity 2017
 
Description WBE for drug use and public health assessment' Amsterdam International Water Week Conference, 30 October - 3 November 2017, Amsterdam 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Talk delivered by Kasprzyk-Hordern entitled: 'WBE for drug use and public health assessment' at Amsterdam International Water Week Conference
Year(s) Of Engagement Activity 2017
 
Description Water fingerprinting for environmental and public health assessment, Centre of Research in Biomedical Science (CRIB), UWE, 4th December 2020 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Talk by Kasprzyk-Hordern focussed on 'Water fingerprinting for environmental and public health assessment', Centre of Research in Biomedical Science (CRIB), UWE, 4th December 2020
Year(s) Of Engagement Activity 2020