LinkPI: Linking Phenotype function with Identity: a novel integrated single-cell technology and metagenomics approach

Lead Research Organisation: University of Glasgow
Department Name: School of Engineering

Abstract

The wide ranging and diverse microorganisms found within the environment play a central role in maintaining sustainability on our planet. However, their ability to function and the functions themselves have been and are being seriously altered by human activities. One key example of this is the development of antimicrobial resistance in wild bacteria as a result of antibiotics that can be found in waste water. The impact of this is a significant emerging threat to the global economy and health. Increasing evidence shows that variations in the genetic makeup of individual cells, together with the way these are manifest in their physical characteristics plays a critical role in the fate of these microorganisms. Despite this knowledge, most studies of microorganisms currently rely on culturing and analysing them as large groups in laboratories, rather than on the individual level. To further complicate matters, the majority of the naturally occurring microbes (>99%) are not capable of being grown in laboratory conditions. This imposes formidable challenges to understand the activities of microbes in situ and their response to the ever-changing environments.

In this project we will establish a novel approach to identify active microbes of interest within complex microbial communities, linking the behaviour and genetic profile of individual cells. Specifically, we will exploit the cutting-edge technology advances to analyse, sort and characterise microbes from a mixture of microorganisms. To achieve this, we will work to develop new devices and protocols for collecting samples on-site. These samples will then be analysed using a range of cutting-edge, lab-based techniques.

One key feature of the project is the forging of new international collaborations with the world leading research groups of complementary expertise. This not only provides us with access to a range of world-class tools, but also enables the local collection and handling of samples from sites of interest around the globe. These include the Yanzi river near Shanghai in China, the River Thames near London and the Han River near Seoul in Korea.

Planned Impact

The outcomes of this work will be wide reaching and of benefit to a range of industrial sectors that actively use or whose work needs to take consideration of microbial communities. These include agriculture, aquaculture, agriculture, environmental biotechnology, wastewater treatment and pharmaceutical industries. The microbial communities used in the processes can play an important role in the spread and evolving of antibiotic resistance. For instance, active sludge in municipal wastewater treatment plants are rich in microbes and has become the "hot spot" hosting antibiotic resistance genes. In addition, these are increasingly concerned about pollution and its impact on antibiotic resistance. This imposes increasing pressure on the antibiotic suppliers - pharmaceutical companies. Our research will contribute to better understanding of these processes and facilitate design of suitable intervention routes. We will plan to engage with these stakeholders from the start and throughout the project. We have long established links with the water treatment sector and the food industry across the UK. We will actively engage with them during the course of this project. They will be invited at the annual Industry Day of the University of Glasgow where we will present our research findings. We will also actively engage in Knowledge Exchange activities which are often organised by the University and will utilise Glasgow Sustainable Development Network to communicate with public and industry.

The research outputs from this work will also be of significance for those involved in legislation and policy. Our methods and tools will contribute to the evidence used by policy makers on which they can develop country-specific action plans for reducing the spread of highly resistance bacterial pathogens. Our team have already engaged with environmental agency in the UK (e.g. Defra, VMD, AZ, Rivers Trust, LGC and AIT). During the project, we will engage with relevant end-users, beneficiaries, stakeholders and policy makers in Asia.

Last, but not least, this work is of societal benefit and the general public are key stakeholders. The data obtained will contribute to the understanding of a wide range of scientific questions of public concern the impact of the spread of antibiotic resistance on food security and health care. As just one example, we will present this work at the Glasgow Science Festival that attracts more than 50,000 visitors each year and we will be on the look out for similar new opportunities that are always arising and being promoted, both in the neighbourhood of the host institutions and further afield.

A blog site detailing our progress will be launched at the beginning of the project. We will ensure that our publications and workshop proceedings are covered by the media and issue press releases throughout the project accordingly. By adopting an open source approach in releasing the software associated with metagenomics on the blog site, we will become a part of a community of users and developers who will have an interest in working together to support each other. Any software bugs will tend to be more visible and more rapidly corrected to improve the software. It will also establish reputation and bring intangible benefits of goodwill.

Publications

10 25 50
 
Description Widespread antibacterial resistance has become a significant threat to global health. A primary route for the spread is via the transmission of antibiotic resistance genes (ARGs) in the environment. Currently, continuously emerging new ARGs requires early identification of antibiotic-resistant bacterial cells. However, naturally occurring microorganisms consist of complex mixtures of individual cells of various phenotypic features. Raman activated cell sorting provides a powerful tool to sort such a complex system and isolate cells according to the characteristic chemical information in Raman spectra. A 3D hydrodynamic flow-focusing device for cell sorting has been recently developed, which substantially improved the robustness and performance of the technology. It will provide a versatile tool for function-based flow cytometry and sorting applications in the fields of microbiology, biotechnology, life science and diagnostics.

With the extension of the project, we have organised a successful international symposium, titled "Single cell technology meets Microbiomics 2021" (https://www.microbiomics2021.org/). The symposium promotes our research. It also provides a forum to show the cutting-edge single-cell technologies and their application in clinical and environmental microbiology, and health care. The high-profile speakers and eye-widening programs attracted 220 participants from 20 countries and enabled new collaborations.
Exploitation Route The Pre-Covid workshops and our recent publications have been pivotal to establishing new collaborations. We have formed a new partnership with Professor Long Nghiem at the University of Technology in Sydney. A review paper on the detection of antibiotic resistance gene and antibiotic bacterial have been prepared during the lock-down and is under revision.
Sectors Agriculture, Food and Drink,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL https://link.springer.com/article/10.1007/s10404-021-02425-y
 
Description We have presented our work on single-cell microfluidics at a number of international conferences and networking events. The technology has attracted new collaborations with scientists in the field of microbiological ecology, industrial biotechnology, environmental microbiology, and wastewater treatment. It also provides a tool for the discovery of new drugs and antibiotic therapies. In collaboration with our current industrial partners, such as Tianjin Modern Innovative Traditional Chinese Medicine (TCM) technology Co. Ltd, we have used our platforms to screen TCMs in the search for effective routes to reduce antibiotic resistance. The technology has also been employed to search for unknown microorganisms from the natural environment, which have the potential to produce new antibiotics.
First Year Of Impact 2022
Sector Agriculture, Food and Drink,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description EPSRC IAA project ""A gut microbiota chip for functional analysis of herbal medicine" co-funders: TianjinTCM and Horiba UK
Amount £108,739 (GBP)
Funding ID 312019/0 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 12/2020 
End 10/2021
 
Description LinkPI: Linking Phenotype function with Identity: a novel integrated single-cell technology and metagenomics approach
Amount £83,396 (GBP)
Funding ID NE/S008721/1 
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 12/2018 
End 11/2020
 
Title Automated Raman based cell sorting system and method 
Description Raman activated cell sorting has emerged as a label-free technology that can link phenotypic function with genotypic properties of cells. We have developed a three-dimensional hydrodynamic focusing microfluidic system for a fully automated, continuous Raman activated cell sorting. It has the ability to sort a range of cell sizes (from 1 µm bacteria to 10's µm mammalian cells) with stable operation over >8 hours and high throughput. 
Type Of Material Improvements to research infrastructure 
Year Produced 2021 
Provided To Others? Yes  
Impact The system provides a versatile tool for function-based flow cytometry and sorting applications in the fields of microbiology, biotechnology, life science and diagnostics. 
 
Description Development of point of care diagnostic test for pathogens in the Mycobacterium tuberculosis complex 
Organisation Bee Robotics Ltd
Country United Kingdom 
Sector Private 
PI Contribution Establish the molecular targets, design of the LAMP primers and establish the approaches to extraction procedures. Defined new loci for targeting detection of pathogen DNA in samples and beta tested specificity and sensitivity of the assay. Tried a range of extraction procedures, confirmed amplicon by minION long read sequencing.
Collaborator Contribution Provision of chemistry expertise, design of housing for the test and approaches to producing field based test.
Impact Multi-disciplinary collaboration still in progress
Start Year 2019
 
Description Environmental reservoirs of microbial pathogens 
Organisation National University of Sciences and Technology
Department Institute of Environmental Sciences and Engineering (IESE)
Country Pakistan 
Sector Academic/University 
PI Contribution Working on developing bids for GCRF and other funding agencies to halt the spread of microbial pathogens and AMR in the environment
Collaborator Contribution Hosted a workshop and paid our team's expenses to visit and work in a collaborative pilot study in addition to talks and visits to field sites
Impact The collaboration resulted in the analysis of field sites to consider the environmental reservoirs of microbial pathogens and understanding transmission pathways through water and food chain routes.
Start Year 2019
 
Description Environmental reservoirs of microbial pathogens 
Organisation University College of Islamabad
Country Pakistan 
Sector Academic/University 
PI Contribution Working on developing bids for GCRF and other funding agencies to halt the spread of microbial pathogens and AMR in the environment
Collaborator Contribution Hosted a workshop and paid our team's expenses to visit and work in a collaborative pilot study in addition to talks and visits to field sites
Impact The collaboration resulted in the analysis of field sites to consider the environmental reservoirs of microbial pathogens and understanding transmission pathways through water and food chain routes.
Start Year 2019
 
Description Korean partnering on metagenomics and microbiome analysis 
Organisation Chung-Ang University
Country Korea, Republic of 
Sector Academic/University 
PI Contribution Understanding the risk posed by the widespread dissemination of antimicrobial resistant bacteria (AMRB) and pathogenic variants (AMRP) in the environment depends on persistence of the bacteria and the resistance genes (ARGs). ARGs can move into indigenous environmental bacteria but risks of infection will be reduced or removed. What has been observed is the high diversity of resistance genotypes in waste water polluted environments coupled with the expansive diversity of the environmental resistome and so the extensive mixing of human and animal wastes with environmental bacteria particularly in the presence of antibiotics could produce AMRP capable of extensive resistance phenotypes. Uncertainty exists over the longevity and activity of AMRP in the environment outside of their hosts and to what extent they may participate in gene exchange so current work in both the Cha and Wellington groups is focused on this aspect and finding new ways of monitoring AMRP and their activities. Thus we identified two major challenges in understanding risk in environmental exposure to AMRP which relates to viability and ARG acquisition. In the form of two workshops and exchange of personnel the Wellington group members Dr Chiara Borsetto and Dr Robert James demonstrated their methods for using long read sequencing to gain an improved analysis of resistance genes in the environmental resistome, Chiara Borsetto talked about using mesocosms to establish if sublethal levels of antibiotics in the environment select for resistance phenotypes or are simply collocated with already resistant bacteria in waste water effluent. By characterizing the host genome it is possible to establish if the resistant bacteria are human adapted or environmental bacteria. From our work on river flumes used to model impact of waste water effluent in UK rivers it appears that sublethal antibiotic has a distinct impact on the prevalence of the relevant resistance genes.
Collaborator Contribution Professor Chang-Jun Cha's group have expertise in bioinformatics and hold a resistance gene database in addition to having a WaferGen SmartChip machine and the arrays which can detect very sensitively over 300 types of resistance genes using PCR conducted in hundreds of minicells.We were given access to this machine and were able to compare results and also establish diversity and new combinations of various mobile genetic elements. We aim to write a position paper comparing our results on anthropogenic impacts on Thames riverine microbiome compared with the study they are conducting on the Han river in South Korea. They provided access to databases and SmartChips plus shared sequences and databases.
Impact Two papers are in progress and one submitted currently: Submission no: ENVINT_2019_218, Submission title: A novel sulfonamide resistance mechanism by two-component flavin-dependent monooxygenase system in sulfonamide-degrading actinobacteria Corresponding author: Professor Chang-Jun Cha, Listed co-author(s): Dr Kihyun Lee, Dr Cung Nawl Thawng, Professor Elizabeth Wellington, Dr Dae-Wi Kim.
Start Year 2018
 
Description Prof Cha 
Organisation Chung-Ang University
Country Korea, Republic of 
Sector Academic/University 
PI Contribution Bring the expertise in microfluidics and single cell analysis, in particular, Raman activated single bacterial cell sorting, and the development of "field-use" devices
Collaborator Contribution Professor Cha will bring the expertise in antimicrobial resistance gene detection from environmental samples using "SmartChip" technology for AMR monitoring and will test the "field-use" devices.
Impact The collaboration is multi-disciplinary, involves engineering, enviromental microbiology and water treatment.
Start Year 2018
 
Description Professor Cheng 
Organisation Boston University
Country United States 
Sector Academic/University 
PI Contribution Microfluidic platforms and Raman activated cell sorting technology
Collaborator Contribution Professor Cheng has been constantly at the most forefront of chemical imaging. He is world-leading in Stimulated Raman Spectroscopy technology. The combination of SRS with our microfluidic sorting platform will enable us to develop the next generation of high throughput cell sorting technology.
Impact The collaboration is multi-disciplinary, involves engineering, microbiology and optical physics.
Start Year 2019
 
Description Professor Ines Mandic Mulec 
Organisation University of Ljubljana
Country Slovenia 
Sector Academic/University 
PI Contribution Provide single-cell technology.
Collaborator Contribution Professor Mandic Mulec is a leading scientist in environmental ecology and has discovered a number of new species. The collaboration between us will significantly enhance our ability to understand the behaviour of these new species as well as their ecological roles in the community.
Impact The collaboration is multi-disciplinary, involves engineering and environmental ecology.
Start Year 2019
 
Description Professor Liu 
Organisation Chinese Academy of Sciences
Department Institute of Microbiology
Country China 
Sector Learned Society 
PI Contribution Bring the expertise in microfluidics and single cell analysis, in particular, Raman activated single bacterial cell sorting, and single-cell microfluidic devices.
Collaborator Contribution Bring expterise in cultivation of microbes from various environments and molecular microbiology techniques.
Impact The collaboration is multi-disciplinary, involves engineering, enviromental microbiology and industrial biotechnology.
Start Year 2018
 
Description TCM 
Organisation Tianjin Modern Innovative Traditional Chinese Medicine Technology Co. Ltd
Country China 
Sector Private 
PI Contribution Provided single-cell microfluidic technology for studying antibiotic resistance of bacteria with single-cell resolution.
Collaborator Contribution The company will bring their expertise in traditional Chinese Medicine (TCM) to the collaboration. Together, we aim to explore the potential of using TCM to reduce the occurrence of antibiotic resistance.
Impact The collaboration is multi-disciplinary, involves engineering, clinical microbiology and pharmaceutical industry.
Start Year 2019
 
Description Univ. Ghent 
Organisation University of Ghent
Department Department of Biochemical and Microbial Technology
Country Belgium 
Sector Academic/University 
PI Contribution Bring the expertise in microfluidics and single cell analysis, in particular, Raman activated single bacterial cell sorting, and the development of "field-use" devices.
Collaborator Contribution Our partner will bring flow cytometry expertise, in particular, a flow cytometric fingerprinting toolbox that can detect phenotypical and phylogenetic changes in microbial communities.
Impact The collaboration is multi-disciplinary, involves engineering, industrial biotechnology and water managment and treatment.
Start Year 2018