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Antimicrobial resistance is an increasing problem on a global scale. Rapid antibiotic susceptibility testing (AST) is urgently needed in the clinic to enable personalized prescriptions in high-resistance environments and to limit the use of broad-spectrum drugs. Current rapid phenotypic AST methods do not include species identification (ID), leaving time-consuming plating or culturing as the only available option when ID is needed to make the sensitivity call. Here we describe a method to perform phenotypic AST at the single-cell level in a microfluidic chip that allows subsequent genotyping by in situ FISH. By stratifying the phenotypic AST response on the species of individual cells, it is possible to determine the susceptibility profile for each species in a mixed sample in 2 h. In this proof-of-principle study, we demonstrate the operation with four antibiotics and mixed samples with combinations of seven species. Rapid antibiotic susceptibility testing (AST) is needed. Here the authors report a method for phenotypic AST at the single cell level, using a microfluidic chip that allows for subsequent genotyping with in situ FISH; they apply this to a mixed sample of 7 species and 4 antibiotics.

The rate at which transcription factors (TFs) bind their cognate sites has long been assumed to be limited by diffusion, and thus independent of binding site sequence. Here, we systematically test this assumption using cell-to-cell variability in gene expression as a window into the in vivo association and dissociation kinetics of the model transcription factor LacI. Using a stochastic model of the relationship between gene expression variability and binding kinetics, we performed single-cell gene expression measurements to infer association and dissociation rates for a set of 35 different LacI binding sites. We found that both association and dissociation rates differed significantly between binding sites, and moreover observed a clear anticorrelation between these rates across varying binding site strengths. These results contradict the long-standing hypothesis that TF binding site strength is primarily dictated by the dissociation rate, but may confer the evolutionary advantage that TFs do not get stuck in near-operator sequences while searching. What makes a transcription factor (TF) binding site “strong”: slow TF dissociation, or fast TF association? Kandavalli et al found that stronger sites had both slower dissociation and faster association compared to weaker sites in vivo.

For effective treatment of bacterial infections, it is essential to identify the species causing the infection as early as possible. Current methods typically require hours of overnight culturing of a bacterial sample and a larger quantity of cells to function effectively. This study uses one-hour phase-contrast time-lapses of single-cell bacterial growth collected from microfluidic chip traps, also known as a “mother machine”. These time-lapses are then used to train deep artificial neural networks (Convolutional Neural Networks and Vision Transformers) to identify the species. We have previously demonstrated this approach on four different species, which is now extended to seven common pathogens causing human infections: Pseudomonas aeruginosa , Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii , Enterococcus faecalis , Proteus mirabilis , and Staphylococcus aureus . Furthermore, we expand upon our previous work by evaluating real-time performance as additional frames are captured during testing, and investigating the role of training set size, data quality, and data augmentation as well as the contribution of texture and morphology to performance. The experiments suggest that spatiotemporal features can be learned from video data of bacterial cell divisions, with both texture and morphology contributing to classifier decision. The method could be used simultaneously with phenotypic antibiotic susceptibility testing (AST) in the microfluidic chip. The best models attained an average precision of 93.5% and a recall of 94.7% (0.997 AUC) on a trap basis in a separate, unseen experiment with mixed species after around one hour. However, in a real-world scenario, one can assume many traps will contain the actual species causing the infection. Still, several challenges remain, such as isolating bacteria directly from blood and validating the method on diverse clinical isolates. This proof of principle study brings us closer to real-time diagnostics that could transform the initial treatment of acute infections.

Reliable detection and classification of bacteria and other pathogens in the human body, animals, food, and water is crucial for improving and safeguarding public health. For instance, identifying the species and its antibiotic susceptibility is vital for effective bacterial infection treatment. Here we show that phase contrast time-lapse microscopy combined with deep learning is sufficient to classify four species of bacteria relevant to human health. The classification is performed on living bacteria and does not require fixation or staining, meaning that the bacterial species can be determined as the bacteria reproduce in a microfluidic device, enabling parallel determination of susceptibility to antibiotics. We assess the performance of convolutional neural networks and vision transformers, where the best model attained a class-average accuracy exceeding 98%. Our successful proof-of-principle results suggest that the methods should be challenged with data covering more species and clinically relevant isolates for future clinical use.

Bacteria evolved genes whose single-cell distributions of expression levels are broad, or even bimodal. Evidence suggests that they might enhance phenotypic diversity for coping with fluctuating environments. We identified seven genes in E. coli with bimodal (low and high) single-cell expression levels under standard growth conditions and studied how their dynamics are modified by environmental and antibiotic stresses known to target gene expression. We found that all genes lose bimodality under some, but not under all, stresses. Also, bimodality can reemerge upon cells returning to standard conditions, which suggests that the genes can switch often between high and low expression rates. As such, these genes could become valuable components of future multi-stable synthetic circuits. Next, we proposed models of bimodal transcription dynamics with realistic parameter values, able to mimic the outcome of the perturbations studied. We explored several models’ tunability and boundaries of parameter values, beyond which it shifts to unimodal dynamics. From the model results, we predict that bimodality is robust, and yet tunable, not only by RNA and protein degradation rates, but also by the fraction of time that promoters remain unavailable for new transcription events. Finally, we show evidence that, although the empirical expression levels are influenced by many factors, the bimodality emerges during transcription initiation, at the promoter regions and, thus, may be evolvable and adaptable.

Cells contain thousands of genes. Many genes are involved in the control of cellular activities. Some activities require a few hundred genes to run largely synchronous transcriptional programs. To achieve this, cells have evolved global regulator (GR) proteins that can influence hundreds of genes simultaneously. We have engineered a library of Escherichia coli strains to track the levels over time of these, phenotypically critical, GRs. Each strain has a single-copy plasmid coding for a fast-maturing green fluorescent protein whose transcription is controlled by a copy of the natural GR promoter. By allowing the tracking of GR levels, with sensitivity and specificity, this library should become of wide use in scientific research on bacterial gene expression (from molecular to synthetic biology) and, later, be used in applications in therapeutics and bioindustries.

Transcription kinetics is limited by its initiation steps, which differ between promoters and with intra- and extracellular conditions. Regulation of these steps allows tuning both the rate and stochasticity of RNA production. We used time-lapse, single-RNA microscopy measurements in live Escherichia coli to study how the rate-limiting steps in initiation of the Plac/ara-1 promoter change with temperature and induction scheme. For this, we compared detailed stochastic models fit to the empirical data in maximum likelihood sense using statistical methods. Using this analysis, we found that temperature affects the rate limiting steps unequally, as nonlinear changes in the closed complex formation suffice to explain the differences in transcription dynamics between conditions. Meanwhile, a similar analysis of the PtetA promoter revealed that it has a different rate limiting step configuration, with temperature regulating different steps. Finally, we used the derived models to explore a possible cause for why the identified steps are preferred as the main cause for behavior modifications with temperature: we find that transcription dynamics is either insensitive or responds reciprocally to changes in the other steps. Our results suggests that different promoters employ different rate limiting step patterns that control not only their rate and variability, but also their sensitivity to environmental changes.

Approximately 50 million people suffer from sepsis yearly, and 13 million die from it. For every hour a patient with septic shock is untreated, their survival rate decreases by 8%. Therefore, rapid detection and antibiotic susceptibility profiling of bacterial agents in the blood of sepsis patients are crucial for determining appropriate treatment. Here, we introduce a method to isolate bacteria from whole blood with high separation efficiency through Smart centrifugation , followed by microfluidic trapping and subsequent detection using deep learning applied to microscopy images. We detected, within 2 h, E. coli , K. pneumoniae , or E. faecalis from spiked samples of healthy human donor blood at clinically relevant concentrations as low as 9, 7 and 32 colony-forming units per ml of blood, respectively. However, the detection of S. aureus remains a challenge. This rapid isolation and detection represents a significant advancement towards culture-free detection of bloodstream infections.

Closely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E . coli . We also studied the sequence (distance between transcription start sites ‘ d TSS ’ , pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and d TSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when d TSS ≤ 35 bp) and RNAP occupancy of the downstream promoter (when d TSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of d TSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of d TSS . Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs.

Temperature shifts trigger genome-wide changes in Escherichia coli's gene expression. We studied if chromosome integration impacts on a gene's sensitivity to these shifts, by comparing the single-RNA production kinetics of a P promoter, when chromosomally-integrated and when single-copy plasmid-borne. At suboptimal temperatures their induction range, fold change, and response to decreasing temperatures are similar. At critically low temperatures, the chromosome-integrated promoter becomes weaker and noisier. Dissection of its initiation kinetics reveals longer lasting states preceding open complex formation, suggesting enhanced supercoiling buildup. Measurements with Gyrase and Topoisomerase I inhibitors suggest hindrance to escape supercoiling buildup at low temperatures. Consistently, similar phenomena occur in energy-depleted cells by DNP at 30 °C. Transient, critically-low temperatures have no long-term consequences, as raising temperature quickly restores transcription rates. We conclude that the chromosomally-integrated P has higher sensitivity to low temperatures, due to longer-lasting super-coiled states. A lesser active, chromosome-integrated native lac is shown to be insensitive to Gyrase overexpression, even at critically low temperatures, indicating that the rate of escaping positive supercoiling buildup is temperature and transcription rate dependent. A genome-wide analysis supports this, since cold-shock genes exhibit atypical supercoiling-sensitivities. This phenomenon might partially explain the temperature-sensitivity of some transcriptional programs of E. coli.