Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is associated with suboptimal glycopeptide treatment outcomes. However, conventional antimicrobial susceptibility testing fails to distinguish hVISA from vancomycin-susceptible Staphylococcus aureus (VSSA), potentially delaying effective therapy. The gold standard population analysis profiling with area under the curve (PAP-AUC) method is labor-intensive and time-consuming, necessitating rapid detection alternatives. This study introduces a microfluidic platform based on fluorescence-activated droplet sorting (FADS) for the rapid detection and isolation of hVISA. Through systematic optimization of key parameters such as droplet generation conditions, bacterial suspension concentration, fluorescence probe selection, fluorescence duration, and vancomycin screening concentration, we established a rapid hVISA screening system. The efficacy of this system was evaluated using the hVISA standard strain Mu3, followed by validation with 15 clinical hVISA isolates. Genomic analysis further elucidated the genetic basis of drug resistance in hVISA strains. We developed a high-throughput detection platform for hVISA by integrating a microfluidic system with FADS. Based on a Poisson distribution theoretical model, the bacterial suspension concentration was optimized to 106 CFU/mL, achieving a single-bacterium droplet encapsulation rate of approximately 30

AbstractStreptomyces is a model bacterium to study multicellular differentiation and the major reservoir for antibiotics discovery. However, the cellular‐level lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools that can mimic their natural conditions. In this study, we developed a simple microfluidic chip for the cultivation and observation of the entire lifecycle of Streptomyces development from the single‐cell perspective. The chip consists of channels for loading samples and supplying nutrients, microwell arrays for the seeding and growth of single spores, and air chambers beside the microwells that facilitate the development of aerial hyphae and spores. A unique feature of this chip is that each microwell is surrounded by a 1.5 µm nanogap connected to an air chamber, which provides a stabilized water–air interface. We used this chip to observe the lifecycle development of Streptomyces coelicolor and Streptomyces griseus germinated from single spores, which revealed differentiation of aerial hyphae with progeny spores at micron‐scale water–air interfaces and air chambers. Finally, we demonstrated the applicability of this chip in phenotypic assays by showing that the microbial hormone A‐Factor is involved in the regulatory pathways of aerial hyphae and spore formation. The microfluidic chip could become a robust tool for studying multicellular differentiation, single‐spore heterogeneity, and secondary metabolism of single‐spore germinated Streptomyces.

Recent characterization of the obligate episymbiont Saccharibacteria (TM7) belonging to the candidate phyla radiation (CPR) has expanded the extent of microbial diversity. However, the episymbiotic lifestyle of TM7 is still underexploited due to the deficiency of cultivated representatives. Here, we describe gene-targeted TM7 cultivation guided by repurposing epicPCR (emulsion, paired isolation, and concatenation PCR) to capture in situ TM7‒host associations. Using this method, we obtained a novel Saccharibacteria isolate TM7i and its host Leucobacter aridicollis J1 from Cicadae Periostracum, the castoff shell of cicada. Genomic analyses and microscopic characterizations revealed that TM7i could bind to J1 through twitching-like motility mediated by type IV pili (T4P). We further showed that the inhibition of T4P extrusion suppressed the motility and host adherence of TM7i, resulting in its reduced growth. However, the inactivation of T4P had little effect on the growth of TM7i that had already adhered to J1, suggesting the essential role of T4P in host recognition by TM7i. By capturing CPR‒host association and elaborating the T4P-dependent episymbiotic association mechanism, our studies shed light on the distinct yet widespread lifestyle of CPR bacteria.

Enzymes that can decompose synthetic plastics such as polyethylene terephthalate (PET) are urgently needed. Still, a bottleneck remains due to a lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation and incubation of droplets encapsulating single cells, picoinjection of fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with this method, including specificity and sensitivity for discriminating PETase from other enzymes. We then optimized its performance and compatibility with environmental samples. The system was used to screen a wastewater sample from a PET textile mill. We successfully obtained PETdegrading species from nine different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate that the FADS pipeline can be widely adopted to discover new plastic-degrading microorganisms and enzymes in various environments and may be utilized in the directed evolution of degrading enzymes using synthetic biology.

ABSTRACTStreptomyces is a model filamentous prokaryote to study multicellular differentiation and a rich reservoir for antibiotics discovery. In their natural conditions, Streptomyces grows at the interface of porous soil, air, and water. The morphological development of Streptomyces is traditionally performed on agar plates and mostly studied at the population levels. However, the detailed lifecycle of Streptomyces has not been well studied due to its complexity and lack of research tools which can mimic their natural conditions in the soil. Here, we developed a simple assembled microfluidic device for cultivation and the entire lifecycle observation of Streptomyces development from single-cell level. The microfluidic device composed of a microchannel for loading samples and supplying nutrients, microwell arrays for seeding and growth of single spores, and air-filled chambers aside of the microwells that facilitate growth of aerial hyphae and spores. A unique feature of this device is that each microwell is surrounded by a 1.5 µm gap connected to an air-filled chamber which provide stabilized water-air interface. We used this device to observe the development of single Streptomyces spores and found that unlike those in bulk liquid culture, Streptomyces can differentiate at water-air interfaces in microscale liquid culture. Finally, we demonstrated that phenotypic A-Factor assay can be performed at defined time point of its lifecycle. This microfluidic device could become a robust tool for studying Streptomyces multi-cellular differentiation and interaction at single cell level.IMPORTANCEWe describe a microfluidic device that mimics the natural porous environment for the growth and development of Streptomyces, the model system for bacterial multicellularity. The microfluidic device is used for cultivation and the entire lifecycle observation of Streptomyces development from single-cell level, including growth of aerial filaments. The aerial hyphae development of Streptomyces at the water-air interface was observed at real time in the microfluidic device. The early growth, opportunistic transformation (in the gap), and merging of aerial hyphae of Streptomyces in the microfluidic device were observed for the first time. It will play an important role in finding single-cell heterogeneity to study secondary metabolites related to the complex lifecycle of Streptomyces.

Candidate phyla radiations (CPR), accounting for a major microbial supergroup with remarkably small genomes and reduced sizes, are widely distributed yet mostly uncultured. Limited culture and its obligate reliance upon other bacteria hindered investigation of their lifestyles. In this work we isolated a CPR bacterium, TM7i, with its host Leucobacter aridocollis J1, by combination of Emulsion, Paired Isolation and Concatenation PCR (epicPCR) detection and filtrate co-culture. Genomic profiling of TM7 genomes and microscopic investigation of TM7i-J1 symbiosis suggest the conservation of type IV pili and a pili-dependent lifestyle of TM7. Further, we observed twitching motility of TM7i mediated by pili and its role played in the interaction with its host. Our results shed a light on the lifestyle about this enigmatic bacterial radiation, which may also be adopted by other CPR organisms. The epicPCR-directed isolation method underlines high efficiency of CPR bacteria isolation and thus may be used in other symbiotic or epibiotic microorganisms.

Microbes thrive and, in turn, influence the earth’s environment, but most are poorly understood because of our limited capacity to reveal their natural diversity and function. Developing novel tools and effective strategies are critical to ease this dilemma and will help to understand their roles in ecology and human health. Recently, droplet microfluidics is emerging as a promising technology for microbial studies with value in microbial cultivating, screening, and sequencing. This review aims to provide an overview of droplet microfluidics techniques for microbial research. First, some critical points or steps in the microfluidic system are introduced, such as droplet stabilization, manipulation, and detection. We then highlight the recent progress of droplet-based methods for microbiological applications, from high-throughput single-cell cultivation, screening to the targeted or whole-genome sequencing of single cells.

Enzymes that can decompose synthetic plastics such as polyethylene terephthalate (PET) are urgently needed. However, a bottleneck remains due to a lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation and incubation of droplets encapsulating single cells, picoinjection of fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with this method, including specificity and sensitivity for discriminating PETase from other enzymes. We then optimized its performance and compatibility with environmental samples. The system was used to screen a wastewater sample from a PET textile mill. We successfully obtained PET-degrading species from nine different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate that the FADS pipeline can be widely adopted to discover new PET-degrading microorganisms and enzymes in various environments and may be utilized in the directed evolution of PETases using synthetic biology.

Droplet microfluidics with picoinjection provides significant advantages to multistep reactions and screenings. The T-junction design for picoinjection is convenient in adding picoliter reagents into passing droplets to initiate reactions. However, conventional picoinjectors face difficulties in eliminating cross-contamination between droplets, preventing them from widespread use in sensitive biological and molecular assays. Here, we introduce stepinjection, which uses a T-junction with a stepped channel design to elevate the diffusional buffer zone into the main channel and consequently increases the pressure difference between droplets and the inlet of the injection channel. To demonstrate the stepinjector's ability to perform contamination-sensitive enzymatic assays, we inject casein fluorescein isothiocyanate (FITC-casein) into a mixture of savinase and savinase-free (labeled with a red fluorescent dye) droplets. We observe no cross-contamination using stepinjection but find a severe cross-talk using an optimal picoinjection design. We envision that the simple, tunable, and reliable stepinjector can be easily integrated in various droplet processing devices, and facilitate various biomedical and biochemical applications including multiplex digital PCR, single-cell sequencing, and enzymatic screening.

Following publication of the original article [1], the authors reported an error in Figure 1 and a text on page 13. In Figure 1, some areas are missing and the correct figure is presented here. On page 13, the sentence should be updated to the following (change has been indicated in bold italics).

The termite gut microbiome is a model system to investigate microbial interactions and their associations with host. For decades, extensive research with molecular tools and conventional cultivation method has been carried out to define the microbial diversity in termite gut. Yet, many bacterial groups of the termite gut microbiome have not been successfully cultivated in laboratory. In this study, we adapted the recently developed microfluidic streak plate (MSP) technique for cultivation of termite gut microbial communities at both aerobic and anaerobic conditions. We found that 99 operational taxonomic units (OTUs) were cultivable by MSP approach and 18 OTUs were documented first time for termite gut microbiota. Further analysis of the bacterial diversities derived by culture-dependent MSP approach and culture-independent 16S rRNA gene typing revealed that both methods have bias in recovery of gut microbiota. In total 396 strains were isolated with MSP technique, and potential new taxa at species and/or genus levels were obtained that were phylogenetically related to Burkholderia, Micrococcus, and Dysgonomonas. Results from this study indicate that MSP technique is applicable for cultivating previously unknown and new microbial groups of termite gut microbiota.

The group of imidazolinone herbicides, widely used for weed control, is hazardous to some sensitive rotational crops. Thus, rapid elimination of imidazolinones from contaminated soil is significant for the environment. Biodegradation studies have demonstrated the ability of chemotaxis to enhance the biodegradation of pollutants. In this study, we used our newly developed SlipChip device for chemotactic sorting and a microfluidic streak plate device for bacterial cultivation as a new pipeline for screening imidazolinone degraders. The degradation efficiencies of an enrichment consortium and a chemotaxis consortium were determined by HPLC-MS/MS. Both consortia degraded all tested imidazolinones, with the highest efficiency (71.8%) for imazethapyr, and the chemotaxis consortium degraded these compounds approximately 10% more efficiently than the enrichment consortium. Moreover, the community diversities of the enrichment consortium and the chemotaxis consortium were analyzed by 16S rRNA gene amplicon sequencing. The results indicated that members of genus Ochrobactrum primarily contribute to the degradation of imidazolinones. This work proved that chemotaxis toward biodegradable pollutants increases their bioavailability and enhances the biodegradation rate. It also provided a new way to screen effective pollutant degraders and can be applied for the selective isolation of other chemotactic species from environmental samples.

Bacterial biofilms play essential roles in biogeochemical cycling, degradation of environmental pollutants, infection diseases, and maintenance of host health. The lack of quantitative methods for growing and characterizing biofilms remains a major challenge in understanding biofilm development. In this study, a dynamic sessile-droplet habitat is introduced, a simple method which cultivates biofilms on micropatterns with diameters of tens to hundreds of micrometers in a microfluidic channel. Nanoliter plugs are utilized, spaced by immiscible carrier oil to initiate and support the growth of an array of biofilms, anchored on and spatially confined to the micropatterns arranged on the bottom surface of the microchannel, while planktonic or dispersal cells are flushed away by shear force of aqueous plugs. The performance of the aforementioned method of cultivating biofilms is demonstrated by Pseudomonas aeruginosa PAO1 and its derived mutants, and quantitative antimicrobial susceptibility testing of PAO1 biofilms. This method could significantly eliminate corner effects, avoid microchannel clogging, and constrain the growth of biofilms for long-term observations. The controllable sessile droplet-based biofilm cultivation presented in this study should shed light on more quantitative and long-term studies of biofilms, and open new avenues for investigation of biofilm attachment, growth, expansion, and eradication.

Regulating mechanisms underlying hepatic myeloid-derived suppressor cell (MDSC) accumulation remain to be described. Here, we provide evidence for the involvement of tumour-activated liver stromal cells in the process of hepatic MDSCs migration and accumulation. Our data showed an elevated frequency of MDSCs in the liver of tumour-bearing mice. Moreover, tumour-activated liver stromal cells promote MDSC migration into the liver site. Further investigation indicated higher levels of cytokine and chemokine expression in liver stromal cells after exposure to the tumour-conditioned supernatant. Notably, the expression levels of proinflammatory factors, mainly including macrophage colony stimulating factor (M-CSF), transforming growth factor-β (TGF-β), monocyte chemotactic protein-1 (MCP-1) and stromal-derived factor-1 (SDF-1), increased after treatment with tumour-conditioned supernatant, and blockade of MCP-1 or SDF-1 decreased the proportion of tumour infiltrated MDSCs in mice co-transplanted with liver stromal cells and tumour cells, but not in mice with only tumour cells injection. These findings demonstrate that tumour-activated liver stromal cells produce higher levels of chemokines and cytokines, which may contribute to MDSC accumulation into the liver site in patients with liver cancer.

Liver dendritic cells (DCs) display immunosuppressive activities and inhibit the CD4+ T cell response. The present study assessed whether and how liver DCs suppress CD8+ T cells. We found that bone marrow-derived mature DCs incubated with liver stromal cells were characterized by a longer life span, reduced CD11c, IA/IE, CD80, CD86, and CD40 expression, and increased CD11b expression. These unique liver stromal cell-educated mature DCs (LSed-DCs) stimulated CD8+ T cells to express CD25 and CD69, but inhibited their proliferation. CD8+ T cell suppression depended on soluble factors released by LSed-DCs, but not cell-cell contact. Compared with mature DCs, LSed-DCs produced more nitric oxide and IL-10. Addition of a nitric oxide synthase inhibitor, PBIT, but not an IL-10-blocking mAb, reversed LSed-DC inhibition of CD8+ T cell proliferation. We also found that LSed-DCs reduced CD8+ T cell-mediated liver damage in a mouse model of autoimmune hepatitis. These results demonstrate that the liver stroma induces mature DCs to differentiate into regulatory DCs that suppress CD8+ T cell proliferation, and thus contribute to liver tolerance.

We report a microfluidic device for automated sorting and cultivation of chemotactic microbes from pure cultures or mixtures. The device consists of two parts: in the first part, a concentration gradient of the chemoeffector was built across the channel for inducing chemotaxis of motile cells; in the second part, chemotactic cells from the sample were separated, and mixed with culture media to form nanoliter droplets for encapsulation, cultivation, enumeration, and recovery of single cells. Chemotactic responses were assessed by imaging and statistical analysis of droplets based on Poisson distribution. An automated procedure was developed for rapid enumeration of droplets with cell growth, following with scale-up cultivation on agar plates. The performance of the device was evaluated by the chemotaxis assays of Escherichia coli (E. coli) RP437 and E. coli RP1616. Moreover, enrichment and isolation of non-labelled Comamonas testosteroni CNB-1 from its 1:10 mixture with E. coli RP437 was demonstrated. The enrichment factor reached 36.7 for CNB-1, based on its distinctive chemotaxis toward 4-hydroxybenzoic acid. We believe that this device can be widely used in chemotaxis studies without necessarily relying on fluorescent labelling, and isolation of functional microbial species from various environments.

Electroencephalographic (EEG)feature values of multiple subjects vary greatly between individual subjects.In light of this,we analyse the influence of data range of single normalisation on classification accuracy.This experiment uses six kinds of common normalisation methods on emotion dataset to compare the accuracies from three kinds of data range in regard to single normalisation.They are the features of all subjects,all features of single subject and the single property feature of single subject.The result proves that the single feature property of single subject is more appropriate as the data range of single normalisation of multiple subjects’EEG data.In addition,we present the feature selection method which combines the variance contribution rate with F-score,it brings a significant reduction in the number of features without reducing the classification accuracies.Wavelet packet tree node energy gets the best classification accuracies as the changed lest feature.The wavelet packet entropy has higher classification accuracies than the EEG rhythm wavelet entropy.

Emerging evidence has suggested that CD8+ T had modulatory function on CD4+ T mediated autoimmune and inflammatory diseases. However, the underlying mechanisms remain unclear. In this study, we found that CD8+ T activation inhibited OVA323–339 antigen specific CD4+ T cells proliferation in vitro and in vivo. Further investigation demonstrated that this immunosuppression largely depended on the soluble factor from activated CD8+ T to modify the phenotype and functions of DCs. Moreover, not only the inhibitors for IDO or iNOS, but also IFN-γ neutralization markedly reversed this immunosuppression on OVA323–339 antigen specific CD4+ T cells proliferation. Interestingly, CD8+ T cells absence aggravated the pathological damage in lung in OVA-induced asthma model, but alleviated by CD8+ T transfer and activation. Thus, these findings suggested that activated CD8+ T population exerted feedback regulation in DCs modification, and then attenuated CD4+ T mediated immune response.

To verify whether the Hilbert-Huang transform (HHT)was appropriate for emotional electroencephalog raphic (EEG)analysis and to get the best feature for classification,multiple features were extracted using Hilbert-Huang transform and wavelet packet transform from emotional EEG data.These features were used to recognition valence.The results showed that the first four IMF components’energy moment percentage and energy percentage in a particular time window could obtain the highest average classification accuracy rate that up to 75%and the latter needed less computation time.Wavelet packet transform needed less time for extracting features than HHT.As a feature,the nodes’energy of wavelet packet decomposition tree’s fifth layer got a classification accuracy rate of 6 9. 7 1%,which was higher than other features extracted by wavelet packet transform. The experiment showed that Hilbert-Huang transform had the advantage of recognition accuracy rate and disadvantage of compu-tation time compared with wavelet packet transform.