Bio-Inspired Fluorescent Carbon Dots as Probes for Rapid Detection of Bacteria in Physiological Samples

Bacterial infections have great public health and economic impact. While at present most can be treated with antibiotics, doing so requires cases of bacterial infections to be recognised early so that they can be treated with the right drugs, while ensuring that antibiotics are not given unnecessarily. With the growth in antibiotic resistance, it is becoming essential that we use these drugs appropriately. At present growth of organisms from patient samples (e.g. urine), a process which takes 18 hours or more, is usually required before specific infecting bacteria can identified. A device able to rapidly detect the presence of bacteria in such samples, and identify which species are present, without this growth step would enable doctors to make rapid and informed decisions about when antibiotic treatment is necessary and which drug should be used.

Here we propose to develop and evaluate a technology for identifying bacteria in patient samples. We will combine a novel series of chemical probes (fluorescent carbon dots, FCDs) that can attach to bacteria to make them fluorescent, with an ultra-sensitive quantum photonic sensor (QPS) developed by our industrial partner, FluoretiQ Ltd., that is able to detect these fluorescent bacteria in patient samples. In order to identify individual species of bacteria we will attach specific sugars (glycans) to the surface of FCDs, exploiting the fact that different bacteria recognise particular sugar molecules as part of the process of binding to the cells of their host. We base our trials around E coli bacteria causing urinary tract infections as these are common conditions that create high workloads for NHS laboratories (our clinical partner processes up to 1000 urine samples per day) and if improperly treated can lead to severe conditions such as sepsis.

We will test this methodology by assessing in the laboratory whether specific bacteria can bind to specific glycan-FCDs. A second series of laboratory experiments will then seek to replicate patient samples by suspending bacteria derived from patients, and cultured human cells, in liquid media designed to mimic the composition of human urine and testing whether glycan-FCDs bind bacteria under these conditions. Finally, with support from clinical microbiologists, we will test whether the glycan-FCD/QPS method can detect and identify bacteria in urine samples from human patients and evaluate its effectiveness compared to methods currently in use. As future users they will also help us to optimise the method and associated instrumentation to ensure that this can be used easily in the clinical laboratory, and provide guidance on how to ensure that our method can be validated against appropriate comparators and demonstrated to comply with NHS quality management systems.

In parallel we will test whether glycan-FCDs can be used as the basis for new treatments for bacterial infections. We have already demonstrated that FCDs can bind to and enter bacteria; preliminary experiments show that they can also kill bacteria, in a light-dependent process. Hence we will investigate whether our modified glycan-FCDs retain the ability to kill bacteria, and whether this killing is specific to the species targeted by the particular surface sugar. We will also attach antibiotics to the surface of FCDs to test whether this represents a method to deliver drugs to specific bacteria, many of which are difficult to kill with antibiotics because the drug is unable to enter the bacterial cell.

The project will establish whether glycan-FCDs can form the basis of a rapid method for detecting infecting bacteria in patient samples in the clinical microbiology laboratory, and whether these can also be used to improve the effectiveness of antibiotics against many of these organisms. In so doing we will also develop new methods for synthesising complex sugar molecules that may be applied in multiple other research areas including drug and vaccine development.

This proposal seeks support to develop a sensitive fluorescence detection technology based on novel glycan-functionalized fluorescent carbon dots (glycan-FCDs) used as probes to label bacteria. In combination with our industrial partner Fluoretiq Ltd.'s Quantum Photonic Sensor (QPS) detection device this will form the basis of a rapid test for the presence of bacteria in clinical samples (e.g. urine), providing clinicians with early warning of infection and informing prescribing decisions. In addition, we will establish whether glycan-FCD nanoplexes can deliver antibiotics to specific pathogens, providing a potential route to expand use of existing antibiotics against Gram-negative bacteria and extend treatment options for these most problematic pathogens. Achieving these aims, and developing this technology into a viable detection device, will provide public health, economic and societal benefits to a variety of stakeholders at regional, national and international levels.

Public health benefits will accrue from reduced impact of bacterial infections resulting from the ability to detect specific bacteria more rapidly than present methodologies, that generally require culturing steps before identification is achieved. Early and appropriate antibiotic administration is key to successful patient outcome; our technology will contribute to reducing morbidity and mortality by providing early identification of infecting bacteria and the requirement for antibiotic treatment. This will improve patient care for a range of conditions by identifying cases when antibiotics should be used and further benefit public health by reducing unnecessary antibiotic prescribing, which has been linked to antimicrobial resistance (AMR). Slowing the rate of resistance emergence will improve treatment outcomes for patients with a wide variety of bacterial infections by prolonging the clinically effective lifetimes of the most useful and cost-effective drugs. This will reduce the numbers of infections requiring more expensive second-line treatments and/or failing to respond to therapy, that generate complications and require additional interventions that increase the burden upon the NHS. In the longer term the ability to deliver antibiotics to Gram-negative bacteria using glycan-FCD nanoplexes will contribute to overcoming AMR by providing additional treatments for these challenging organisms.

Economic benefit will result from the impact of activities around device design, manufacture and marketing; and more widely from a reduced economic burden of bacterial infections. A successful outcome to this project will be a functioning device able to detect key bacterial species in clinical specimens (e.g. urine samples) with comparable sensitivity and specificity to existing methods but operating on a much shorter time scale. This will engage additional partners (clinical end users and investors) to support larger scale device production and more extensive trials, enabling the industrial partner (FluoretiQ) to expand their operations, creating skilled jobs associated with refining the device, validating its effectiveness in the clinical laboratory; and ultimately manufacture, sales and marketing. While the total cost of bacterial infections to the UK economy is difficult to calculate; it is clear that by reducing the infection burden our technology will have substantial economic impact.

A reduction in incidence of bacterial infections achieved by successful introduction of rapid bacterial identification and more targeted antimicrobial therapeutics will also carry significant societal benefits, as patients will recover sooner and their prognosis will be improved both physically and emotionally, improving their ability to contribute socially and/or economically. Reducing the numbers of microbial infections will thus reduce their impact upon the lives of patients' families and social and professional circles, as well as upon the patients themselves.