Proceedings of the NASA Microbiology Workshop

Long-term spaceflight is characterized by extraordinary challenges to maintain the life-supporting instrumentation free from microbial contamination and the crew healthy. The methodology currently employed for microbial monitoring in space stations or short spaceflights within the orbit of Earth have been instrumental in safeguarding the success of the missions, but suffers certain shortcomings that are critical for long spaceflights. This workshop addressed current practices and methodologies for microbial monitoring in space systems, and identified and discussed promising alternative methodologies and cutting-edge technologies for pursuit in the microbial monitoring that hold promise for supporting future NASA long-duration space missions

Determination of antibiotic susceptibility of the bacteria causing urinary tract infections using a novel lab-on-a-chip design

Urinary tract infections (UTIs) are one of the most common types of bacterial infection in the UK, and also are expensive to treat costing the National Health Service ~£54 million between 2016 and 2017. Culture-based antibiotic susceptibility testing (AST) is used to identify an antibiotic to treat drug-resistant urinary tract infections and takes 48 hours to complete. Faster prescription of effective antibiotics should reduce the risk of sepsis and poor clinical outcomes. To address this need, we developed a Lab-on-a-Chip (LOC) based method to conduct electrochemical AST using screen-printed macroelectrodes (SPEs) and antibiotic-loaded hydrogels. SPEs were fabricated using carbon-graphite based inks, with resazurin bulk modified SPEs (R-SPEs) being fabricated through modification of the SPEs WE. Polyvinyl alcohol (PVA) based hydrogels were loaded with the following antibiotics were used; cephalexin, ceftriaxone, colistin, gentamicin, piperacillin, trimethoprim and vancomycin as well as an antibiotic-free control. LOC devices were then designed to encapsulate both the R-SPEs and the antibiotic hydrogels to enable multiplexed electrochemical AST to occur on a single device. In the initial testing of the R-SPEs and the antibiotic hydrogels independently of a LOC device, antibiotic susceptibility could be determined in 90 minutes for E. coli. After the preliminary work, eight chambered LOC devices were spiked with simulated UTI samples. Each chamber contained an R-SPE and an antibiotic hydrogel. After an incubation step, susceptibility of Escherichia coli and Klebsiella pneumoniae could be established in 85 minutes of testing which is significantly faster than the 48 hours required for conventional culture-based AST. The sensitive detection of resazurin afforded by using the electrochemical detection methodology incorporated onto a LOC device described here offers an inexpensive and simple method for the determination of antibiotic susceptibility that is faster than using a culture-based approach

Testing Anti-Biofilm Polymeric Surfaces : Where to Start?

Present day awareness of biofilm colonization on polymeric surfaces has prompted the scientific community to develop an ever-increasing number of new materials with anti-biofilm features. However, compared to the large amount of work put into discovering potent biofilm inhibitors, only a small number of papers deal with their validation, a critical step in the translation of research into practical applications. This is due to the lack of standardized testing methods and/or of well-controlled in vivo studies that show biofilm prevention on polymeric surfaces; furthermore, there has been little correlation with the reduced incidence of material deterioration. Here an overview of the most common methods for studying biofilms and for testing the anti-biofilm properties of new surfaces is provided

Switchable lanthanide fluorescence probes in homogenous molecular diagnostics

The number of molecular diagnostic assays has increased tremendously in recent years.Nucleic acid diagnostic assays have been developed, especially for the detection of human pathogenic microbes and genetic markers predisposing to certain diseases. Closed-tube methods are preferred because they are usually faster and easier to perform than heterogenous methods and in addition, target nucleic acids are commonly amplified leading to risk of contamination of the following reactions by the amplification product if the reactions are opened. The present study introduces a new closed-tube switchable complementation probes based PCR assay concept where two non-fluorescent probes form a fluorescent lanthanide chelate complex in the presence of the target DNA. In this dual-probe PCR assay method one oligonucleotide probe carries a non-fluorescent lanthanide chelate and another probe a light absorbing antenna ligand. The fluorescent lanthanide chelate complex is formed only when the non-fluorescent probes are hybridized to adjacent positions into the target DNA bringing the reporter moieties in close proximity. The complex is formed by self-assembled lanthanide chelate complementation where the antenna ligand is coordinated to the lanthanide ion captured in the chelate. The complementation probes based assays with time-resolved fluorescence measurement showed low background signal level and hence, relatively high nucleic acid detection sensitivity (low picomolar target concentration). Different lanthanide chelate structures were explored and a new cyclic seven dentate lanthanide chelate was found suitable for complementation probe method. It was also found to resist relatively high PCR reaction temperatures, which was essential for the PCR assay applications. A seven-dentate chelate with two unoccupied coordination sites must be used instead of a more stable eight- or nine-dentate chelate because the antenna ligand needs to be coordinated to the free coordination sites of the lanthanide ion. The previously used linear seven-dentate lanthanide chelate was found to be unstable in PCR conditions and hence, the new cyclic chelate was needed. The complementation probe PCR assay method showed high signal-to-background ratio up to 300 due to a low background fluorescence level and the results (threshold cycles) in real-time PCR were reached approximately 6 amplification cycles earlier compared to the commonly used FRET-based closed-tube PCR method. The suitability of the complementation probe method for different nucleic acid assay applications was studied. 1) A duplex complementation probe C. trachomatis PCR assay with a simple 10-minute urine sample preparation was developed to study suitability of the method for clinical diagnostics. The performance of the C. trachomatis assay was equal to the commercial C. trachomatis nucleic acid amplification assay containing more complex sample preparation based on DNA extraction. 2) A PCR assay for the detection of HLA-DQA1*05 allele, that is used to predict the risk of type 1 diabetes, was developed to study the performance of the method in genotyping. A simple blood sample preparation was used where the nucleic acids were released from dried blood sample punches using high temperature and alkaline reaction conditions. The complementation probe HLA-DQA1*05 PCR assay showed good genotyping performance correlating 100% with the routinely used heterogenous reference assay. 3) To study the suitability of the complementation probe method for direct measurement of the target organism, e.g., in the culture media, the complementation probes were applied to amplificationfree closed-tube bacteriophage quantification by measuring M13 bacteriophage ssDNA. A low picomolar bacteriophage concentration was detected in a rapid 20- minute assay. The assay provides a quick and reliable alternative to the commonly used and relatively unreliable UV-photometry and time-consuming culture based bacteriophage detection methods and indicates that the method could also be used for direct measurement of other micro-organisms. The complementation probe PCR method has a low background signal level leading to a high signal-to-background ratio and relatively sensitive nucleic acid detection. The method is compatible with simple sample preparation and it was shown to tolerate residues of urine, blood, bacteria and bacterial culture media. The common trend in nucleic acid diagnostics is to create easy-to-use assays suitable for rapid near patient analysis. The complementation probe PCR assays with a brief sample preparation should be relatively easy to automate and hence, would allow the development of highperformance nucleic acid amplification assays with a short overall assay time.Siirretty Doriast

A portable metabolomics-on-CMOS platform for point-of-care testing

Metabolomics is the study of the metabolites, small molecules produced during the metabolism. Metabolite levels mirror the health status of an individual and therefore have enormous potential in medical point-of-care (POC) applications. POC platforms are miniaturised and portable systems integrating all steps from sample collection to result of a medical test. POC devices offer the possibility to reduce the diagnostic costs, shorten the testing time, and, ultimately, save lives for several applications. The glucose meter, arguably the most successful example of metabolomics POC platform, has already demonstrated the dramatic impact that such platforms can have on the society. Nevertheless, other relevant metabolomic tests are still relegated to centralised laboratories and bulky equipment. In this work, a metabolomics POC platform for multi-metabolite quantification was developed. The platform aims to untap metabolomics for the general population. As case studies, the platform was designed and evaluated for prostate cancer and ischemic stroke. For prostate cancer, new affordable diagnostic tools to be used in conjunction with the current clinical standard have are needed to reduce the medical costs due to overdiagnosis and increase the survival rate. Thus, a novel potential metabolic test based on L-type amino acids (LAA) profile, glutamate, choline, and sarcosine blood concentrations was developed. For ischemic stroke, where the portable and rapid test can make a difference between life and death, lactate and creatinine blood levels were chosen as potential biomarkers. All the target metabolites were quantified using an optical method (colorimetry). The platform is composed of three units: the cartridge, the reader, and the graphical user interface (GUI). The cartridge is the core of the platform. It integrates a CMOS 16x16 array of photodiodes, capillary microfluidics, and biological receptors onto the same ceramic package. To measure multiple metabolites, a novel method involving a combination of replica moulding and injection moulding was developed for the monolithic integration of microfluidics onto integrated chips. The reader is composed of a custom PCB and a microcontroller board. It is used for addressing, data digitisation and data transfer to the GUI. The GUI - a software running on a portable electronic device - is used for interfacing the system, visualise, acquire, process, and store the data. The analysis of the microfluidic structures showed successful integration. The selection of the specific chemistry for detecting the analytes of interest was demonstrated to be suitable for the performance of the sensors. Quick and reliably capillary flow of human plasma, serum and blood was demonstrated. On-chip quantification of the target metabolites was demonstrated in diluted human serum and human plasma. Calibration curves, kinetics parameter and other relevant metrics were determined. For all the metabolites, the limits of detection were lower than the physiological range, demonstrating the capability of the platform to be used in the target applications. Multi-metabolite testing capability was also demonstrated using commercially and clinically sourced human plasma. For multiplexed assays, reagents were preloaded in the microfluidic channel and lyophilised. Lyophilisation also improved the shelf-life of the reagents. Alternative configurations, involving the use of paper microfluidics, integration of passive blood filter and use of whole blood, were investigated. The chracterisation of the platform culminated with a clinical evaluation for both the target applications. The same platform with minimal modification of the cartridge was able to provide clinically relevant information for both the distinct applications, highlighting the versatility of the platform for POC determination of metabolic biomarkers. For prostate cancer, the platform was used for the quantification of the potential metabolic biomarker in 10 healthy samples and 16 patients affected by prostate cancer. LAA, glutamate and choline average concentrations were elevated in the cancer group with respect to the control group and were therefore regarded as metabolic biomarkers in this population. Metabolomic profiles were used to train a classifier algorithm, which improved the performance of the current clinical blood test, for this population. For ischemic stroke, lactate determination was performed in clinically sourced samples. Clinical evaluation for ischemic stroke was performed using 10 samples from people diagnosed with ischemic stroke. Results showed that the developed platform provided comparable results with an NHS-based gold standard method in this population. This comparison demonstrated the potential of the platform for its on-the-spot use. The developed platform has the potential to lead the way to a new generation of low-cost and rapid POC devices for the early and improved diagnosis of deadly diseases

Biosensors for Diagnosis and Monitoring

Biosensor technologies have received a great amount of interest in recent decades, and this has especially been the case in recent years due to the health alert caused by the COVID-19 pandemic. The sensor platform market has grown in recent decades, and the COVID-19 outbreak has led to an increase in the demand for home diagnostics and point-of-care systems. With the evolution of biosensor technology towards portable platforms with a lower cost on-site analysis and a rapid selective and sensitive response, a larger market has opened up for this technology. The evolution of biosensor systems has the opportunity to change classic analysis towards real-time and in situ detection systems, with platforms such as point-of-care and wearables as well as implantable sensors to decentralize chemical and biological analysis, thus reducing industrial and medical costs. This book is dedicated to all the research related to biosensor technologies. Reviews, perspective articles, and research articles in different biosensing areas such as wearable sensors, point-of-care platforms, and pathogen detection for biomedical applications as well as environmental monitoring will introduce the reader to these relevant topics. This book is aimed at scientists and professionals working in the field of biosensors and also provides essential knowledge for students who want to enter the field

Light-Activated Antimicrobial Surfaces Containing Quantum Dots for the Prevention of Hospital-Acquired Infections

This thesis details the development of effective light-activated antimicrobial polymers for use in healthcare environments, with the aim of reducing hospital-acquired infections (HAIs). The overuse and misuse of antibiotics is the most important factor that has led to increased incidence of multi-drug resistant HAIs. In the hospital setting where there is an abundance of immunosuppressed patients and often hygiene protocols are not strictly followed, HAIs can spread quickly, leading to increased length of hospital stay, morbidity and mortality and high healthcare costs. Self-disinfecting surfaces can reduce the incidence of HAIs by reducing the levels of bacteria on frequently touched hospital surfaces that serve as bacterial reservoirs, thus reducing the risk of HAI transmission. Quantum dots (QDs), extremely small nanoparticles that exhibit unique size-dependent properties, combined with photosensitisers display potent strong bactericidal activity upon incorporation into polymer surfaces. When irradiated under ambient white light, polymer surfaces induce the lethal photosensitisation of a range of Gram-positive and Gram-negative bacteria through the production of reactive oxygen species (ROS). ROS cause irreversible damage leading to cell apoptosis and death by attacking bacterial cells in a non-specific fashion thus making the development of resistance unlikely. Polyurethane substrates were impregnated with QDs and photosensitiser dye (crystal violet) using a modified version of the simple and easily scalable dipping procedure known as the “swell-encapsulation-shrink” technique. Solely cadmium-free, indium-based QDs were used in this study, thereby circumventing issues regarding toxicity arising from the release of cadmium ions from traditional, commonly prepared QDs such CdTe, CdSe and CdS. Materials were characterised using techniques such as UV-Vis absorbance spectroscopy, fluorescence spectroscopy and transmission electron microscopy. The prepared polymer substrates were activated under white light conditions mimicking those used in the hospital (~500 – 6000 lux). In order to deduce the photochemical pathway responsible for light-activated antibacterial activity, whether Type I, Type II or both, the antimicrobial surfaces were tested in a series of microbiological assays using specific ROS inhibitors and quenchers. The surfaces were tested against a range of nosocomial pathogens including Escherichia coli, Staphylococcus aureus, epidemic methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. The novel materials described in this thesis demonstrate very strong self-disinfecting properties even under low light levels, demonstrating their potential for use in hospitals to reduce HAIs without the use of antibiotics