A pico-injection-aided digital droplet detection platform is presented that integrates loop-mediated isothermal amplification (LAMP) with molecular beacons (MBs) for the ultrasensitive and quantitative identification of pathogens, leveraging the sequence-specific detection capabilities of MBs. The microfluidic device contained three distinct functional units including droplet generation, pico-injection, and droplet counting. Utilizing a pico-injector, MBs are introduced into each droplet to specifically identify LAMP amplification products, thereby overcoming issues related to temperature incompatibility. Our methodology has been validated through the quantitative detection of Escherichia coli, achieving a detection limit as low as 9 copies/μL in a model plasmid containing the malB gene and 3 CFU/μL in a spiked milk sample. The total analysis time was less than 1.5 h. The sensitivity and robustness of this platform further demonstrated the potential for rapid pathogen detection and diagnosis, particularly when integrated with cutting-edge microfluidic technologies.

IncX3 plasmids carrying the New Delhi metallo-β-lactamase-encoding gene, blaNDM-5, are rapidly spreading globally in both humans and animals. Given that carbapenems are listed on the WHO AWaRe watch group and are prohibited for use in animals, the drivers for the successful dissemination of Carbapenem-Resistant Enterobacterales (CRE) carrying blaNDM-5-IncX3 plasmids still remain unknown. We observe that E. coli carrying blaNDM-5-IncX3 can persist in chicken intestines either under the administration of amoxicillin, one of the largest veterinary β-lactams used in livestock, or without any antibiotic pressure. We therefore characterise the blaNDM-5-IncX3 plasmid and identify a transcription regulator, VirBR, that binds to the promoter of the regulator gene actX enhancing the transcription of Type IV secretion systems (T4SS); thereby, promoting conjugation of IncX3 plasmids, increasing pili adhesion capacity and enhancing the colonisation of blaNDM-5-IncX3 transconjugants in animal digestive tracts. Our mechanistic and in-vivo studies identify VirBR as a major factor in the successful spread of blaNDM-5-IncX3 across one-health AMR sectors. Furthermore, VirBR enhances the plasmid conjugation and T4SS expression by the presence of copper and zinc ions, thereby having profound ramifications on the use of universal animal feeds.

The increasing prevalence of drug-resistant Escherichia coli (E. coli) resulting from the excessive utilization of antibiotics necessitates the immediate exploration of alternative approaches to counteract pathogenic E. coli. Phages, with their unique antibacterial mechanisms, are considered promising candidates for treating bacterial infections. Herein, we isolated a lytic Escherichia phage Tequatrovirus YZ2 (phage YZ2), which belongs to the genus Tequatrovirus. The genome of phage YZ2 consists of 168,356 base pairs with a G + C content of 35.34% and 269 putative open reading frames (ORFs). Of these, 146 ORFs have been annotated as functional proteins associated with nucleotide metabolism, structure, transcription, DNA replication, translation, and lysis. In the mouse model of a skin wound infected by E. coli, phage YZ2 therapy significantly promoted the wound healing. Furthermore, histopathological analysis revealed reductions in IL-1β and TNF-α and increased VEGF levels, indicating the potential of phages as effective antimicrobial agents against E. coli infection.

Vitamin B5 [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel \"PEACES\" system by (1) expressing the NAD+ kinase gene ppnk from Clostridium glutamicum and the NADP+-dependent gapCcae from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.

Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis.

Inferring gene regulatory network (GRN) is one of the important challenges in systems biology, and many outstanding computational methods have been proposed; however there remains some challenges especially in real datasets. In this study, we propose Directed Graph Convolutional neural network-based method for GRN inference (DGCGRN). To better understand and process the directed graph structure data of GRN, a directed graph convolutional neural network is conducted which retains the structural information of the directed graph while also making full use of neighbor node features. The local augmentation strategy is adopted in graph neural network to solve the problem of poor prediction accuracy caused by a large number of low-degree nodes in GRN. In addition, for real data such as E.coli, sequence features are obtained by extracting hidden features using Bi-GRU and calculating the statistical physicochemical characteristics of gene sequence. At the training stage, a dynamic update strategy is used to convert the obtained edge prediction scores into edge weights to guide the subsequent training process of the model. The results on synthetic benchmark datasets and real datasets show that the prediction performance of DGCGRN is significantly better than existing models. Furthermore, the case studies on bladder uroepithelial carcinoma and lung cancer cells also illustrate the performance of the proposed model.

This study aims to explore the essential functional requirements associated with controlling the proliferation of microbes in the domain of textiles used in public health areas. Herein, three antimicrobial agents, specifically iodopropylbutylcarbamate (IPBC), 1-hydroxypyridine-2-thioketone zinc (ZPT), and 2-octyl-3-isothiazolinone (OIT), were chosen for fabric finishing based on their notable effectiveness, minimal toxicity, cost-efficiency, and chemical stability. Utilizing Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) as representative bacterial strains, the Minimum Inhibitory Concentration (MIC50) of individual and combined antimicrobial agents was measured, and their antimicrobial effectiveness was rigorously evaluated. Concurrently, the antimicrobial effectiveness, whiteness, and mechanical durability of the fabric following antimicrobial treatment were thoroughly examined. The results demonstrate that some combinations of the three antimicrobial agents elicit additive effects on both S. aureus and E. coli. Notably, at an equivalent ratio of IPBC, ZPT, and OIT and a total concentration of 0.2 wt. %, the inhibition rates against both bacterial strains surpass 99%. Upon application to nylon fabric, the treated material demonstrates significant antimicrobial properties, with minimal reduction observed in the whiteness and tensile strength of the treated nylon. This study provides practicable strategies relevant to the production of textiles endowed with antimicrobial properties.

ZnO nanorod nonwoven fabrics (ZNRN) were developed through hydrothermal synthesis to facilitate the prevention of the transmission of respiratory pathogens. The superhydrophobicity and antibacterial properties of ZNRN were improved through the response surface methodology. The synthesized material exhibited significant water repellency, indicated by a water contact angle of 163.9°, and thus demonstrated antibacterial rates of 91.8% for Escherichia coli (E. coli) and 79.75% for Staphylococcus aureus (S. aureus). This indicated that E. coli with thinner peptidoglycan may be more easily killed than S. aureus. This study identified significant effects of synthesis conditions on the antibacterial effectiveness, with comprehensive multivariate analyses elucidating the underlying correlations. In addition, the ZnO nanorod structure of ZNRN was characterized through SEM and XRD analyses. It endows the properties of superhydrophobicity (thus preventing bacteria from adhering to the ZNRN surface) and antibacterial capacity (thus damaging cells through the puncturing of these nanorods). Consequently, the alignment of two such features is desired to help support the development of personal protective equipment, which assists in avoiding the spread of respiratory infections.

The phosphoenol pyruvate-oxaloacetate-pyruvate-derived amino acids (POP-AAs) comprise native intermediates in cellular metabolism, within which the phosphoenol pyruvate-oxaloacetate-pyruvate (POP) node is the switch point among the major metabolic pathways existing in most living organisms. POP-AAs have widespread applications in the nutrition, food, and pharmaceutical industries. These amino acids have been predominantly produced in Escherichia coli and Corynebacterium glutamicum through microbial fermentation. With the rapid increase in market requirements, along with the global food shortage situation, the industrial production capacity of these two bacteria has encountered two bottlenecks: low product conversion efficiency and high cost of raw materials. Aiming to push forward the update and upgrade of engineered strains with higher yield and productivity, this paper presents a comprehensive summarization of the fundamental strategy of metabolic engineering techniques around phosphoenol pyruvate-oxaloacetate-pyruvate node for POP-AA production, including L-tryptophan, L-tyrosine, L-phenylalanine, L-valine, L-lysine, L-threonine, and L-isoleucine. Novel heterologous routes and regulation methods regarding the carbon flux redistribution in the POP node and the formation of amino acids should be taken into consideration to improve POP-AA production to approach maximum theoretical values. Furthermore, an outlook for future strategies of low-cost feedstock and energy utilization for developing amino acid overproducers is proposed.

The spatial organization of bacterial chromosomes is crucial for cellular functions. It remains unclear how bacterial chromosomes adapt to high-temperature stress. This study delves into the 3D genome architecture and transcriptomic responses of Escherichia coli under heat-stress conditions to unravel the intricate interplay between the chromosome structure and environmental cues. By examining the role of macrodomains, chromosome interaction domains (CIDs), and nucleoid-associated proteins (NAPs), this work unveils the dynamic changes in chromosome conformation and gene expression patterns induced by high-temperature stress. It was observed that, under heat stress, the short-range interaction frequency of the chromosomes decreased, while the long-range interaction frequency of the Ter macrodomain increased. Furthermore, two metrics, namely, Global Compactness (GC) and Local Compactness (LC), were devised to measure and compare the compactness of the chromosomes based on their 3D structure models. The findings in this work shed light on the molecular mechanisms underlying thermal adaptation and chromosomal organization in bacterial cells, offering valuable insights into the complex inter-relationships between environmental stimuli and genomic responses.