A Guide to Biodetection in Droplets.

Droplet-based methods for optical biodetection enable unprecedented high-throughput experimental parameters. The methods, however, remain underused due to the accompanying multidisciplinary and complicated experimental workflows. Here, we provide a tutorial for droplet-based optical biodetection workflows with a focus on the key aspect of label selection. By discussing and guiding readers through recent state-of-the-art studies, we aim to make droplet-based approaches more accessible to the general scientific public.

Replacement of milk fat by rapeseed oil stabilised emulsion in commercial yogurt.

The incorporation of lipid droplets and further characterization of matrices within dairy products may be possible using such adjacent particles as protein complexes/lipids. Among the range of varied emulsions and their functionalities, great attention has recently focused on the fabrication of high internal phase types. Feasibly, stable alternatives structured with health-beneficial lipids like those derived from plants could replace saturated fatty acids. As a fat replacement strategy, the fate of incorporated HIPE would require some adjustments either with storage stability and/or structural feat for the food matrix. Therefore, the replacement of milk fat by rapeseed oil stabilised emulsion in commercial yogurt was investigated. This involved 25%, 50% and 75% rapeseed oil respectively assigned as low (LIPE), medium (MIPE), and high internal phase emulsion (HIPE). Specifically, emulsions were examined by droplet size, encapsulation, pH, zeta potential, phase separation, and rheology. The fat free yogurt supplemented by HIPE were examined by droplet size, zeta potential, pH, color, sensory, texture and microbiological aspects against positive (regular milk fat) and negative (fat free) yogurt controls. Results showed increasing rapeseed oil contents would form smaller droplet-like emulsions. Within the yogurt matrix however, incorporating HIPE would seemingly reduce oil droplet size without much compromise to bacterial viability, sensory, or texture. Overall, this simple method of lipid alternation shows promise in dairy products.

User-friendly analysis of droplet array images.

Water-in-oil droplets allow performing massive experimental parallelization and high-throughput studies, such as single-cell experiments. However, analyzing such vast arrays of droplets usually requires advanced expertise and sophisticated workflow tools, which limits accessibility for a wider user base in the fields of chemistry and biology. Thus, there is a need for more user-friendly tools for droplet analysis. In this article, we deliver a set of analytical pipelines for user-friendly analysis of typical scenarios in droplet experiments. We built pipelines that combine various open-source image-analysis software with a custom-developed data processing tool called "EasyFlow". Our pipelines are applicable to the typical experimental scenarios that users encounter when working with droplets: i) mono- and polydisperse droplets, ii) brightfield and fluorescent images, iii) droplet and object detection, iv) signal profile of droplets and objects (e.g., fluorescence).

Droplet-based methods for tackling antimicrobial resistance.

Application of droplet-based methods enables (i) faster detection, (ii) increased sensitivity, (iii) characterization of the level of heterogeneity in response to antibiotics by bacterial populations, and (iv) expanded screening of the effectiveness of antibiotic combinations. Hereby, we discuss the key steps and parameters of droplet-based experiments to investigate antimicrobial resistance. We also review recent findings accomplished with these methods and highlight their advantages and capacity to yield new insights into the problem of antimicrobial resistance.

Cyclohexanohemicucurbit[8]uril Inclusion Complexes With Heterocycles and Selective Extraction of Sulfur Compounds From Water.

Solid-phase extraction that utilizes selective macrocyclic receptors can serve as a useful tool for removal of chemical wastes. Hemicucurbiturils are known to form inclusion complexes with suitably sized anions; however, their use in selective binding of non-charged species is still very limited. In this study, we found that cyclohexanohemicucurbit[8]uril encapsulates five- and six-membered sulfur- and oxygen-containing unsubstituted heterocycles, which is investigated by single-crystal X-ray diffraction, NMR spectroscopy, isothermal titration calorimetry, and thermogravimetry. The macrocycle acts as a promising selective sorption material for the extraction of sulfur heterocycles, such as 1,3-dithiolane and α-lipoic acid, from water.

Investigation of Different Free Image Analysis Software for High-Throughput Droplet Detection.

Droplet microfluidics has revealed innovative strategies in biology and chemistry. This advancement has delivered novel quantification methods, such as droplet digital polymerase chain reaction (ddPCR) and an antibiotic heteroresistance analysis tool. For droplet analysis, researchers often use image-based detection techniques. Unfortunately, the analysis of images may require specific tools or programming skills to produce the expected results. In order to address the issue, we explore the potential use of standalone freely available software to perform image-based droplet detection. We select the four most popular software and classify them into rule-based and machine learning-based types after assessing the software's modules. We test and evaluate the software's (i) ability to detect droplets, (ii) accuracy and precision, and (iii) overall components and supporting material. In our experimental setting, we find that the rule-based type of software is better suited for image-based droplet detection. The rule-based type of software also has a simpler workflow or pipeline, especially aimed for non-experienced users. In our case, CellProfiler (CP) offers the most user-friendly experience for both single image and batch processing analyses.

Techniques Used for Analyzing Microplastics, Antimicrobial Resistance and Microbial Community Composition: A Mini-Review.

Antimicrobial resistance (AMR) is a global health threat. Antibiotics, heavy metals, and microplastics are environmental pollutants that together potentially have a positive synergetic effect on the development, persistence, transport, and ecology of antibiotic resistant bacteria in the environment. To evaluate this, a wide array of experimental methods would be needed to quantify the occurrence of antibiotics, heavy metals, and microplastics as well as associated microbial communities in the natural environment. In this mini-review, we outline the current technologies used to characterize microplastics based ecosystems termed "plastisphere" and their AMR promoting elements (antibiotics, heavy metals, and microbial inhabitants) and highlight emerging technologies that could be useful for systems-level investigations of AMR in the plastisphere.

Epidemiology of Danish Aeromonas salmonicida subsp. salmonicida in Fish Farms Using Whole Genome Sequencing.

Furunculosis, a serious infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida is common in sea-reared rainbow trout production in Denmark. Developing an effective control strategy requires knowledge of the epidemiology, as well as the genomic and virulent variability of the Danish A. salmonicida subsp. salmonicida isolates. To obtain this, the genomes of 101 A. salmonicida subsp. salmonicida, including 99 Danish isolates, one Scottish strain and the type strain NCIMB 1102, were sequenced using the Illumina HiSeq platform. Isolates were de novo assembled, examined for presence of plasmids, virulence and iron acquisition proteins, genomic islands, and antibiotic resistance genes. Single Nucleotide Polymorphisms were aligned and subjected to Bayesian temporal phylogenetic and maximum likelihood tree reconstruction using the published genome of A. salmonicida subsp. salmonicida A449 as reference. Bayesian temporal phylogenetic reconstruction suggests that four major introductions of A. salmonicida subsp. salmonicida into Denmark have occurred. The introductions correlate with the freshwater and subsequent seawater expansion of rainbow trout production. Initial transmission of the bacterium could have been from seawater to freshwater or vice versa, and most minor clades include a mixture of strains from different fresh- and seawater farms. Genomic variation of A. salmonicida subsp. salmonicida mostly appeared to be associated with their plasmids and plasmid encoded virulence factors. Nine A. salmonicida subsp. salmonicida isolates harbored worldwide known antibiotic resistance genes against several antibiotics and there is an indication that 33% of the isolates contained the genomic island AsaGEI1b. These findings not only support the usefulness of whole genome sequencing for genetic studies of homogeneous bacteria in general, but provide novel information about the Danish A. salmonicida subsp. salmonicida population, with implications for vaccine development in efforts to better protect Danish rainbow trout in the future.

Co-infection with 'Candidatus Neoehrlichia Mikurensis' and Borrelia afzelii in Ixodes ricinus ticks in southern Sweden.

The tick-borne bacterium 'Candidatus Neoehrlichia mikurensis' has recently been recognized as a human pathogen in Europe and appears to be the second most common pathogenic bacterium in Ixodes ricinus ticks in central Europe, second to Borrelia afzelii. Here, we investigate the prevalence of 'Candidatus N. mikurensis' in host-seeking ticks in southern Sweden and the rate of co-infection with B. afzelii. We developed a real-time qPCR assay targeting the groEL gene of 'Candidatus N. mikurensis' and applied this assay to 949 I. ricinus ticks collected at several locations over 2 years. We found an overall prevalence of 6.0%, which means that Candidatus N. mikurensis is one of the most common tick-transmitted zoonotic agents in this area. Co-infections with both 'Candidatus N. mikurensis' and B. afzelii occurred in 2.1% of the ticks, which is significantly more than expected under random co-occurrence. The infection intensity (number of bacterial cells) of 'Candidatus N. mikurensis' was not affected by co-infection with B. afzelii, and vice versa. We conclude that there is a risk for simultaneous transmission of these 2 tick-borne pathogens. The potential medical consequences of this require further investigation.