Edgar D. Goluch

BACKGROUND: Urine glucose dipstick tests have low sensitivity, particularly in detecting glucose in early or mild cases of diabetes in cats and dogs. Ketone dipstick tests only detect acetoacetate and do not measure β-hydroxybutyrate (BHB) which is the predominant ketone in diabetic patients and the most reliable indicator of diabetic ketoacidosis (DKA). OBJECTIVE: To evaluate the accuracy, sensitivity, and specificity of urine dipstick tests for glucose and ketone measurements in cats and dogs. To assess whether blood-based Fora meters provide more reliable urine measurements for diabetes detection. METHODS: Standard solutions of glucose, BHB, and acetoacetate in artificial urine were analyzed using Fora meters and dipstick tests. Urine samples from 37 random dogs and 27 random cats were tested and results were analyzed using regression analysis, confusion matrices, and Bland-Altman plots. RESULTS: Fora meters accurately detected glucose and BHP ketone levels in artificial urine with strong linearity to standards. Urine dipstick tests showed moderate accuracy (74%) but low sensitivity (57%) for glucose; performance varied by species and specific gravity. Urine dipstick test underestimate specific gravity in samples with high pH or high glucose levels. Urine dipstick tests cannot be used for BHP ketone measurements (sensitivity < 1%). CLINICAL RELEVANCE: Fora meters even though originally designed for blood, may offer a more precise, point-of-care alternative to dipsticks for diagnosing diabetes and DKA in cats and dogs. Improved urine glucose and ketone quantification can help reduce false diagnosis and support earlier and more accurate treatment decisions.

We tested the feasibility of a novel machined silicon nanopore enrichment device to recover individual microbial taxa from anaerobic sediments. Unlike other environmental isolation devices that have multiple entry points for bacteria or require the sample to be manually placed inside of a culturing chamber, our silicon device contains 24 precisely sized and spaced nanopores, each of which is connected to one culturing well, thereby providing only one entry point for bacteria. The culturing wells allow nutrient transport, so the bacteria that enter continue to experience their natural chemical environment, allowing collection of microbes without manipulating the environment. The device was deployed in marsh sediment and subsequently returned to the laboratory for bacterial culturing and analysis. 16S rRNA marker gene and metagenomic sequencing was used to quantify the number of different microbial taxa cultured from the device. The 16S rRNA sequencing results indicate that each well of the device contained between 1 and 62 different organisms from several taxonomic groups, including likely novel taxa. We also sequenced the metagenome from 8 of the 24 wells, enabling the reconstruction of 56 metagenomic assembled genomes (MAGs), and 44 of these MAGs represented non-redundant genome reconstructions. These results demonstrate that our novel silicon nanofluidic device can be used for isolating and culturing consortia containing a small number of microbial taxa from anaerobic sediments, which can be very valuable in determining their physiological potential. Importance There are very few methods that can remove a few bacterial cells from a complex environment and keep the cells alive so that they can propagate sufficiently to be analyzed in a laboratory. Such methods are important to develop because the physiological functions of individual species of bacteria are often unknown, cannot be determined directly in the complex sample, and many bacterial cells cannot be grown outside of their natural environment. A novel bacterial isolation device has been made tested in a salt marsh. The results show that the device successfully isolated small groups of bacterial species from the incredibly diverse surroundings. The communities of bacteria were easily removed from the device in the laboratory and analyzed.

We present the design and characterization of a low cost, thread-based electrophoretic device with integrated electrochemical detection. The device has an end-channel pencil graphite electrode placement system for performing electrochemical detection on the thread electrophoresis platform with direct sample pipetting onto the thread. We also established the use of methylene blue and neutral red as a pair of reference migration markers for separation techniques coupled with electrochemical detection, as they have different colors for visual analysis and are both electroactive. Importantly, neutral red was also found to migrate at a similar rate to the EOF, indicating that it can be used as a visual identifier of EOF. The utility of our system was demonstrated by electrophoretic separation and electrochemical detection of physiologically relevant concentrations of pyocyanin in a solution containing multiple electroactive compounds. Pyocyanin is a biomarker for the detection of pathogenic Pseudomonas aeruginosa and has a redox potential that is similar to that of methylene blue. The system was able to effectively resolve methylene blue, neutral red, and pyocyanin in less than 7 min of electrophoretic separation. The theoretical limit of detection for pyocyanin was determined to be 559 nM. The electrophoretic mobilities of methylene blue (0.0236 ± 0.0007 mm²/V·s), neutral red (0.0149 ± 0.0007 mm²/V·s), and pyocyanin (0.0107 ± 0.0003 mm²/V·s) were also determined.

One of the hallmark characteristics of chronic diabetic wounds is the presence of biofilm‐forming bacteria. Bacteria encapsulated in a biofilm may coexist as a polymicrobial community and communicate with each other through a phenomenon termed quorum sensing (QS). Here, we describe the QS circuits of bacterial species commonly found in chronic diabetic wounds. QS relies on diffusion of signaling molecules and the local concentration changes of these molecules that bacteria experience in wounds. These biochemical signaling pathways play a role not only in biofilm formation and virulence, but also in wound healing. They are, therefore, key to understanding the distinctive nature of these infections. While several in vivo and in vitro models exist to study QS in wounds, there has been limited progress in understanding the interplay between quorum sensing molecules and host factors that contribute to wound healing. Lastly, we examine the potential of targeting QS for both diagnosis and therapeutic intervention purposes.

We report the design and operation of an integrated microfluidics system that uses cellulose ester dialysis membranes coupled with disposable carbon and copper electrodes for monitoring and concentration of microliter scale biofluid samples. Dialysis membranes are typically used for buffer exchange but in this work, membranes with 100 – 500 dalton MWCO were evaluated for feasibility in concentrating small volume samples. This is an alternative to the use of centrifugation, ultrafiltration, and evaporative methods where quantitative inline monitoring of sample concentration is not feasible. The impact of draw solution used, osmotic concentration gradient, pH and temperature were studied for the optimized concentration of bodily fluids. The system using sucrose generated better results, with water removal rates of 0.023 mL min-1. PBS, urine, and saliva samples were concentrated up to twenty fold (PBS), fifteen fold (urine), and five-fold (saliva) in less than 3 hours. The osmotic system further showed a five-fold increase in the electrochemical signal for detecting pyocyanin, a biomarker for early diagnostics of the Pseudomonas aeruginosa pathogen in urine and saliva samples. Overall, the osmotic system can be easily integrated with point of care diagnostic systems for low cost improvement in signal amplification and limit of detection.

This review provides a comprehensive summary of issues associated with treating polyclonal bacterial biofilms in chronic diabetic wounds. We use this as a foundation and discuss the alternatives to conventional antibiotics and the emerging need for suitable drug delivery systems.

Advances in next-generation sequencing technology along with decreasing costs now allow the microbial population, or microbiome, of a location to be determined relatively quickly. This research reveals that microbial communities are more diverse and complex than ever imagined. New and specialized instrumentation is required to investigate, with high spatial and temporal resolution, the dynamic biochemical environment that is created by microbes, which allows them to exist in every corner of the Earth. This review describes how electrochemical probes and techniques are being used and optimized to learn about microbial communities. Described approaches include voltammetry, electrochemical impedance spectroscopy, scanning electrochemical microscopy, separation techniques coupled with electrochemical detection, and arrays of complementary metal-oxide-semiconductor circuits. Microbial communities also interact with and influence their surroundings; therefore, the review also includes a discussion of how electrochemical probes optimized for microbial analysis are utilized in healthcare diagnostics and environmental monitoring applications.

A major cause of hospital‐acquired infections is Pseudomonas aeruginosa , a gram‐negative bacterial pathogen. This bacterium secretes a unique, redox‐active molecule known as pyocyanin that can serve as a useful biomarker for identification and detection of this opportunistic pathogen. This current study investigates the impact to pyocyanin production and ability to electrochemically distinguish pyocyanin when P. aeruginosa is co‐cultured with other bacterial pathogens. To accomplish this, P. aeruginosa was co‐cultured with up to four different clinically‐relevant bacteria typically found in a wound infection. P. aeruginosa ’s production of pyocyanin was electrochemically monitored over a three day period and the results demonstrated that pyocyanin can be readily measured and that there were no observed enhancements or inhibitory effects to the production of pyocyanin. This information is valuable in the development of this sensing platform for clinical point‐of‐care diagnostics, where polymicrobial growth at the site of infection is common.