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Water-in-oil droplet microfluidics promises capacity for high-throughput single-cell antimicrobial susceptibility assays and investigation of drug resistance mechanisms. Every droplet must serve as an isolated environment with a controlled antibiotic concentration in such assays. While technologies for generation, incubation, screening, and sorting droplets mature, predictable retention of active molecules inside droplets remains a major outstanding challenge. Here, we analyzed 36 descriptors of the antibiotic molecules against experimental results on the cross-talk of antibiotics in droplets. We show that partition coefficient and fractional polar surface area are the key physicochemical properties that predict antibiotic retention. We verified the prediction by monitoring growth inhibition by antibiotic-loaded neighboring droplets. Our experiments also demonstrate that transfer of antibiotics between droplets is concentration- and distance-dependent. Our findings immediately apply to designing droplet antibiotic assays and give deeper insight into the retention of small molecules in water-in-oil emulsions.

We present work of our COST Action on "Understanding and exploiting the impacts of low pH on micro-organisms". First, we summarise a workshop held at the European Federation of Biotechnology meeting on Microbial Stress Responses (online in 2020) on "Industrial applications of low pH stress on microbial bio-based production", as an example of an initiative fostering links between pure and applied research. We report the outcomes of a small survey on the challenging topic of developing links between researchers working in academia and industry that show that, while people in different sectors strongly support such links, barriers remain that obstruct this process. We present the thoughts of an expert panel held as part of the workshop above, where people with experience of collaboration between academia and industry shared ideas on how to develop and maintain links. Access to relevant information is essential for research in all sectors, and because of this we have developed, as part of our COST Action goals, two resources for the free use of all researchers with interests in any aspects of microbial responses to low pH. These are (1) a comprehensive database of references in the literature on different aspects of acid stress responses in different bacterial and fungal species, and (2) a database of research expertise across our network. We invite the community of researchers working in this field to take advantage of these resources to identify relevant literature and opportunities for establishing collaborations.

Water-in-oil droplets allow researchers to perform massive experimental parallelization and high-throughput studies, such as single-cell experiments. However, the analysis of such vast arrays of droplets usually requires advanced expertise and sophisticated workflow tools, which limits the accessibility for wider user base in 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-based experiments. We build the pipelines combining different open-source image-analysis software with the custom-developed data visualization tool “EasyFlow”. Our pipelines are designed to be applicable for the typical experimental scenarios users encounter 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).

Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations.

Droplet microfluidic assays are rapidly gaining popularity because they enable analysis of biochemical reactions, individual cells or small cell populations with high sensitivity, precision and accuracy in a high-throughput manner. Nonetheless, there is a demand for user-friendly and low-cost droplet analysis technology. In this article, we meet this demand by developing two droplet analysis pipelines via free open-source software CellProfiler (CP) and its companion (CellProfiler Analyst, CPA). To illustrate the competence of the pipelines as an independent, and freely accessible droplet analysis tool for any researcher without the need of programming skills, we show (i) droplet digital quantification of viable fluorescent bacteria using single-color images and (ii) analysis of a multi-color fluorescence image with droplets containing different chemical compositions.

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.

The yield of product (cheese) during the cheese-making process depends on the cutting time of the cheese curd. However, the determination of optimal cutting time on an industrial scale is difficult as current standard methods are destructive or analyse only small volumes and not the entire milk to be curdled into cheese. This paper presents a novel front-face fluorimeter (FFF) that is designed to be immersed into a milk batch to enable the determination of the cutting time of cheese curd without the destruction of the sample. The FFF sensor signal corresponds to physical changes in milk during cheese formation and has high predictive power (r &gt, 0.85) and good accuracy (RSE = 30%, considering daily variation between milk samples). The performance of the presented fluorimeter was on par with standard rheological and Berridge methods.

High-throughput microflow cytometry has become a focal point of research in recent years. In particular, droplet microflow cytometry (DMFC) enables the analysis of cells reacting to different stimuli in chemical isolation due to each droplet acting as an isolated microreactor. Furthermore, at high flow rates, the droplets allow massive parallelization, further increasing the throughput of droplets. However, this novel methodology poses unique challenges related to commonly used fluorometry and fluorescent microscopy techniques. We review the optical sensor technology and light sources applicable to DMFC, as well as analyze the challenges and advantages of each option, primarily focusing on electronics. An analysis of low-cost and/or sufficiently compact systems that can be incorporated into portable devices is also presented.

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.

Solid-state electrodes have gained attention in past decades for application in pH sensing as a miniature and mechanically stable alternative to conventional fragile glass electrodes. As a material for solid-state electrodes ruthenium oxide has shown the best characteristics. In this study we investigated the sensitivity and stability of RuO2 screen printed electrodes, produced from the commercially available conductive paste. To enhance the performance, electrodes were coated with Nafion protective layer. Influence of the concentration of Nafion coating solution, drop-casted on the sensitive area of the electrode, was investigated. RuO 2 pH electrodes covered with Nafion layer have shown super- Nernstian response and longer response time, which come into agreement with the result published by other authors previously.

Microbes from the three domains of life, Bacteria, Archaea, and Eukarya, share the need to sense and respond to changes in the external and internal concentrations of protons. When the proton concentration is high, acidic conditions prevail and cells must respond appropriately to ensure that macromolecules and metabolic processes are sufficiently protected to sustain life. While, we have learned much in recent decades about the mechanisms that microbes use to cope with acid, including the unique challenges presented by organic acids, there is still much to be gained from developing a deeper understanding of the effects and responses to acid in microbes. In this perspective article, we survey the key molecular mechanisms known to be important for microbial survival during acid stress and discuss how this knowledge might be relevant to microbe-based applications and processes that are consequential for humans. We discuss the research approaches that have been taken to investigate the problem and highlight promising new avenues. We discuss the influence of acid on pathogens during the course of infections and highlight the potential of using organic acids in treatments for some types of infection. We explore the influence of acid stress on photosynthetic microbes, and on biotechnological and industrial processes, including those needed to produce organic acids. We highlight the importance of understanding acid stress in controlling spoilage and pathogenic microbes in the food chain. Finally, we invite colleagues with an interest in microbial responses to low pH to participate in the EU-funded COST Action network called EuroMicropH and contribute to a comprehensive database of literature on this topic that we are making publicly available. info:eu-repo/semantics/publishedVersion

Academic microfluidics has decisively shifted in recent years from the research on phenomenology and proof-of-concept fluidic functionalities to the developments oriented at applications with biology, medicine and biotechnology in prime focus. Significant efforts are made to demonstrate that microfluidics can be used in unspecialized laboratories to perform previously mundane tasks faster and easier, or to venture into new research areas that were unavailable or unattractive when only classical means of microbiology or biotechnology were employed. Here we review a variety of biological experiments recently performed in microfluidic assays. We categorize the microfluidic systems by the key role they play in the biological experiments as: (i) controlled reaction chambers, (ii) high-throughput arrays, or (iii) micro-positioning systems. We also discuss the outlook for further development and applications of microfluidics in biological sciences.

Measurement of antibiotic susceptibility at the level of single cells is important as it reveals the concentration of an antibiotic that leads to drug resistance in bacterial strains. To date, no solution for large-scale studies of antibiotic susceptibility at the single-cell level has been shown. Here, we present a method for production and separation of emulsions consisting of subnanoliter droplets that allows us to identify each emulsion by their spatial position in the train of emulsions without chemical barcoding. The emulsions of droplets are separated by a third immiscible phase, thus forming large compartments-tankers-each filled with an emulsion of droplet reactors. Each tanker in a train can be set under different reaction conditions for hundreds or thousands of replications of the same reaction. The tankers allow for long term incubation - needed to check for growth of bacteria under a screen of conditions. We use microfluidic tankers to analyze susceptibility to cefotaxime in ca. 1900 replications for each concentration of the antibiotic in one experiment. We test cefotaxime susceptibility for different initial concentrations of bacteria, showing the inoculum effect down to the level of single cells for more than a hundred single-cell events per tanker. Lastly, we use tankers to observe the formation of aggregates of bacteria in the presence of cefotaxime in the increasing concentration of the antibiotic. The microfluidic tankers allow for facile studies of the inoculum effect and antibiotic susceptibility, and constitute an attractive, label-free screening method for a variety of other experiments in chemistry and biology.

Droplet microfluidic assays are rapidly gaining popularity as the result of the ability to manipulate and monitor single biological molecules, individual cells or small populations of bacteria in pico- and nanoliter droplets, with high sensitivity, precision and accuracy in a high-throughput manner. Nonetheless, there is a demand for user-friendly and low-cost droplet analysis technology. In this article we meet this demand with free open-source software CellProfiler (CP). To illustrate the competence of CP as a droplet analysis tool, we show droplet digital quantification of viable fluorescent bacteria.

This paper proves that dodecylresorufin (C12R) outperforms resorufin (the conventional form of this dye) in droplet microfluidic bacterial assays. Resorufin is a marker dye that is widely used in different fields of microbiology and has increasingly been applied in droplet microfluidic assays and experiments. The main concern associated with resorufin in droplet-based systems is dye leakage into the oil phase and neighboring droplets. The leakage decreases the performance of assays because it causes averaging of the signal between the positive (bacteria-containing) and negative (empty) droplets. Here we show that C12R is a promising alternative to conventional resorufin because it maintains higher sensitivity, specificity, and signal-to-noise ratio over time. These characteristics make C12R a suitable reagent for droplet digital assays and for monitoring of microbial growth in droplets.

Here we demonstrate a direct droplet digital PCR assay (dddPCR) for simultaneous identification and quantification of bacteria in a bacterial mixture. We encapsulate bacterial cells in droplets together with PCR reagents to amplify specific target genes. Our method introduces accurate and precise quantitation of bacteria in a bacterial mixture by digital PCR, while eliminating the errors and workload associated with extraction and purification of DNA. The method thus combines the precision and specificity of digital PCR with robustness of a direct PCR. We also show that dddPCR is more tolerant to inhibition by non-specific bacteria as compared to traditional direct real-time PCR.

Heteroresistance is a phenomenon where isogenic bacteria population exhibits a diverse antibiotic resistance pattern at sub-population or single cell level. The sub-populations with higher resistance can remain undetected with conventional diagnostics which makes them subsequently harder to treat. Such surviving phenotypically heterogeneous sub-populations are also a potential hotbed for novel mutations, thus increasing the resistance permanently in bacteria. Droplet microfluidics gives tools for high-throughput analysis of bacteria and their response to antibiotics at single cell level, which is difficult to obtain with traditional agar plate technologies. In here we show for the first time the precise digital quantification of drug resistance profile in isogenic population at single cell level. We also see that the inhibiting amount of drug per bacteria remains quite stable regardless of bacteria density. Interestingly, the bacteria clump together preferably near these sub-inhibitory conditions. The technology and findings we describe here provide novel quantitative insight into the heteroresistance which is a key step in understanding the pathways leading to drug resistance. This knowledge is crucial in the context of global drug resistance threat as it can help us to find tools to prevent further escalation of drug resistance.

Standard digital assays need a large number of compartments for precise quantification of a sample over a broad dynamic range. We address this issue with an optimized droplet digital approach that uses a drastically reduced number of compartments for quantification. We generate serial logarithmic dilutions of an initial bacterial sample as an array of microliter-sized droplet plugs. In a subsequent step, these droplets are split into libraries of nanoliter droplets and pooled together for incubation and analysis. We show that our technology is at par with traditional dilution plate count for quantification of bacteria, but has the advantage of simplifying the experimental setup and reducing the manual workload. The method also has the potential to reduce the assay time significantly.

There is a growing need for quick and reliable methods for microorganism detection and identification worldwide. Although traditional culture-based technologies are trustworthy and accurate at a relatively low cost, they are also time- and labor-consuming and are limited to culturable bacteria. Those weaknesses have created a necessity for alternative technologies that are capable for faster and more precise bacterial identification from medical, food or environmental samples. The most common current approach is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to the specific nucleic acid profiles that are present. This review aims to give an up-to-date overview of different nucleic acid target sequences and respective analytical technologies.

Droplet microfluidics has rapidly emerged as one of the key technologies opening up new experimental possibilities in microbiology. The ability to generate, manipulate and monitor droplets carrying single cells or small populations of bacteria in a highly parallel and high throughput manner creates new approaches for solving problems in diagnostics and for research on bacterial evolution. This review presents applications of droplet microfluidics in various fields of microbiology: i) detection and identification of pathogens, ii) antibiotic susceptibility testing, iii) studies of microbial physiology and iv) biotechnological selection and improvement of strains. We also list the challenges in the dynamically developing field and new potential uses of droplets in microbiology.

We present a trans -translation mediating tmRNA molecule from bacteria that can be used as a marker in microbial diagnostics and/or taxonomical studies. Different bacteria can be detected and specifically identified using surface bound complementary DNA oligonucleotide probes on different sensor platforms. Several tmRNA hybridization techniques were studied on microarray and in real-time on SOI microring resonator biosensor platforms. Microarray based detection technology enabled detection of tmRNA molecules from less than 1 cell equivalent of pathogenic bacteria.

A label-free biosensing method for the sensitive detection and identification of bacterial transfer-messenger RNA (tmRNA) is presented employing arrays of silicon photonic microring resonators. Species specific tmRNA molecules are targeted by complementary DNA capture probes that are covalently attached to the sensor surface. Specific hybridization is monitored in near real-time by observing the resonance wavelength shift of each individual microring. The sensitivity of the biosensing platform allowed for detection down to 53 fmol of Streptococcus pneumoniae tmRNA, equivalent to approximately 3.16 × 107 CFU of bacteria. The simplicity and scalability of this biosensing approach makes it a promising tool for the rapid, PCR-free identification of different bacteria via tmRNA profiling.

Background We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity. Results We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. Conclusions The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

Background Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. Results Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. Conclusion We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology.

This paper describes a diagnostics strategy involving sample preparation, isothermal Nucleic Acid Sequence Based Amplification (NASBA) of bacterial tmRNA, fluorescent labeling, and microarray hybridisation to detect and identify Streptococcus pneumoniae at a concentration of less than one colony forming unit (CFU) per ml of culture. The tmRNA transcript of the bacterial ssrA gene exhibits several properties that make it suitable as a molecular target for nucleic acid diagnostics. Sequence homology at the 5' and 3' ends of the gene facilitates universal amplification of tmRNA from different species, while sequence heterogeneity in the internal portions of the gene permit the design of oligonucleotide probes to identify bacteria at the genus and species level. When combined with the universal amplification of tmRNA and the potential high probe density of microarrays, the method presented here may have application for the high-throughput screening of samples for a multitude of pathogenic bacteria.

We report on a novel label-free biosensor for in situ measurements in liquid. It is comprised of a porous carbon nanotube (CNT) fibre attached to one prong of a quartz tuning fork (QTF) resonator. Only the protruding CNT fibre is immersed into a liquid, while the QTF is kept above the liquid to avoid short circuit of its electrodes. The low density and large surface area of the CNT fibre assure sufficient sensitivity without affecting the performance of the QTF significantly. Efficiency of the sensor was demonstrated experimentally by comparison of the adsorption rate of bovine serum albumin to the CNT fibre at two different pH values; differences in adsorption rates were clearly distinguishable.

Background The hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34°C resulting in low signal intensities. Results We demonstrate that adding specific DNA helper oligonucleotides (chaperones) to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of Streptococcus pneumoniae tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46°C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule. Conclusions The sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.