Double-layer Method Research Articles

Characterization and genome analysis of lytic Vibrio phage VPK8 with potential in lysing Vibrio parahaemolyticus isolates from clinical and seafood sources

BackgroundVibrio parahaemolyticus is a marine bacterium causing seafood-associated gastrointestinal illness in humans and acute hepatopancreatic necrosis disease (AHPND) in shrimp. Bacteriophages have emerged as promising biocontrol agents against V. parahaemolyticus. This study characterizes Vibrio phage VPK8, focusing on host specificity, efficiency of plating (EOP) variability across V. parahaemolyticus isolates from diverse sources and other Vibrio species, morphology, genomic features, and bacteriolytic potential.MethodsVibrio phage VPK8 was isolated from blood cockles in Thailand using a mixed-host approach and purified via the double-layer agar method. Host specificity was evaluated using spot assays and EOP measurements against 120 Vibrio strains, including AHPND-associated, clinical, and seafood isolates. Phage morphology was characterized by transmission electron microscopy (TEM), while genomic features were analyzed using next-generation sequencing. Lytic characteristics, including latent period and burst size, were determined through one-step growth curves, and bacterial growth reduction was evaluated over a 24-h.ResultsVibrio phage VPK8 is a lytic phage with a 42,866 bp linear double-stranded genome, G + C content of 49.4%, and 48 coding sequences. Phylogenetic analysis grouped it within the Autographiviridae family, showing 95.96% similarity to Vibrio phage vB_VpaP_MGD1. Viral proteomic analysis placed VPK8 within the Pseudomonadota host group. Spot assays indicated broad lytic activity, but EOP analysis revealed high infectivity in clinical and seafood V. parahaemolyticus isolates, as well as some V. cholerae and V. mimicus strains. TEM revealed an icosahedral head (~ 60 nm) and a short tail. At a multiplicity of infection of 0.01, VPK8 exhibited a latent period of 25 min, a burst size of 115, and effectively inhibited the reference host V. parahaemolyticus PSU5124 within 6 h, maintaining its lytic activity and stability for over 24 h.ConclusionsThis study provides a detailed characterization of Vibrio phage VPK8 which exhibits targeted infectivity with high EOP in clinical and seafood V. parahaemolyticus isolates, as well as selected Vibrio species. Its stable lytic performance, rapid replication, and genomic safety suggest its potential for phage-based applications. Further studies should explore its in vivo efficacy and the genetic features contributing to phage resistance mechanisms, enhancing its potential applicability in managing Vibrio-related diseases.

Biological Characteristics and Whole-Genome Analysis of a Porcine E. coli Phage.

(1) Background: In recent years, the increasing emergence of multidrug-resistant pathogens in pig farms has begun to pose a severe threat to animal welfare and, by extension, public health. In this study, we aimed to explore the biological characteristics and genomic features of bacteriophages that are capable of lysing porcine multidrug-resistant E. coli, which was isolated from sewage. In doing so, we provided a reference for phage therapies that can be used to treat multidrug-resistant strains. (2) Method: Using the multidrug-resistant E. coli isolate sq-1 as the host bacterium, bacteriophages were isolated and purified from fecal samples using a double-layer agar plate method. The morphology was observed using a transmission electron microscope, and its host range, optimal multiplicity of infection (MOI), one-step growth curve, thermal stability, acid-base tolerance, and in vitro antibacterial ability were tested. Genomic features were analyzed using whole-genome sequencing. (3) Results: A lytic phage named vB_EcoS_Psq-1 (abbreviated as Psq-1) was successfully isolated. Electron microscopy revealed that Psq-1 belongs to the family of long-tailed phages, possessing clear and transparent plaques of approximately 1 mm in diameter. Psq-1 only lyses the host bacterium and does not affect other E. coli strains or other species of bacteria. The optimal MOI for phage Psq-1 was 0.1, with a latent period of 25 min, an exponential growth period of 25 min, and a lysis yield of 44.21 PFU/cell. Its activity remains stable at temperatures between 40 °C and 60 °C and from pH 4.0 to pH 13.0. Psq-1 exhibited a significant inhibitory effect on E. coli in liquid culture medium. The nucleic acid type of phage Psq-1 was dsDNA, with a total genome length of 44,183 bp and a GC content of 52.16%. No known resistance, lysogenic, or virulence-related genes were detected. The whole genome contains 55 open reading frames (ORFs). (4) Conclusions: This study isolated a bacteriophage that is capable of lysing multidrug-resistant E. coli. Characterized by a narrow E. coli lysis range, a long latent period, limited lytic ability, and stable biological properties, this bacteriophage can serve as a reference isolate for E. coli phages and can provide biological materials and data to support research on bacteriophages that are effective against multidrug-resistant porcine E. coli.

Optimizing the production and efficacy of antimicrobial bioactive compounds from Streptomyces kanamyceticus in combating multi-drug-resistant pathogens.

The rise of antibiotic-resistant pathogens has intensified the search for novel antimicrobial agents. This study aimed to isolate Streptomyces kanamyceticus from local soil samples and evaluate its antimicrobial properties, along with optimizing the production of bioactive compounds. Soil samples were collected from local regions, processed, and analysed for Streptomyces strains isolation using morphological characteristics and molecular identification through 16S rRNA gene PCR assay. Antimicrobial activity was assessed against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Candida albicans using the double-layer method, while Minimum Inhibitory Concentration (MIC) values were determined. The extracted compounds underwent Fourier Transform Infrared Spectroscopy (FTIR) analysis for functional group identification. Optimization of bioactive compound production was performed using a Central Composite Design (CCD) coupled with Partial Least Squares Regression (PLSR). A total of 25 distinct Streptomyces strains were isolated, with seven confirmed as S. kanamyceticus. These strains exhibited antimicrobial activity, with inhibition zones reaching 30 mm and MIC values between 20 and 70 µg/mL. The extraction yielded 150-200 mL of bioactive compounds. Optimization studies revealed that a medium containing 10 g/L glucose and 10 g/L glycine max meal maximized antibiotic production. This study confirmed that S. kanamyceticus is a promising source of novel antibiotics. The combination of microbial isolation, antimicrobial testing, and statistical optimization successfully enhanced the production of bioactive compounds, contributing to the search for effective antimicrobial agents against resistant pathogens.

Standardization of the Agar Plate Method for Bacteriophage Production.

The growing threat of antimicrobial resistance (AMR), exacerbated by the COVID-19 pandemic, highlights the urgent need for alternative treatments such as bacteriophage (phage) therapy. Phage therapy offers a targeted approach to combat bacterial infections, particularly those resistant to conventional antibiotics. This study aimed to standardize an agar plate method for high-mix, low-volume phage production, suitable for personalized phage therapy. Plaque assays were conducted with the double-layer agar method, and plaque sizes were precisely measured using image analysis tools. Regression models developed with Minitab software established correlations between plaque size and phage production, optimizing production while minimizing resistance development. The resulting Plaque Size Calculation (PSC) model accurately correlated plaque size with inoculum concentration and phage yield, establishing specific plaque-forming unit (PFU) thresholds for optimal production. Using phages targeting pathogens such as Escherichia, Salmonella, Staphylococcus, Pseudomonas, Chryseobacterium, Vibrio, Erwinia, and Aeromonas confirmed the model's accuracy across various conditions. The model's validation showed a strong inverse correlation between plaque size and minimum-lawn cell clearing PFUs (MCPs; R² = 98.91%) and identified an optimal inoculum density that maximizes yield while minimizing the evolution of resistant mutants. These results highlight that the PSC model offers a standardized and scalable method for efficient phage production, which is crucial for personalized therapy and AMR management. Furthermore, its adaptability across different conditions and phages positions it as a potential standard tool for rapid and precise phage screening and propagation in both clinical and industrial settings.

Modeling and analysis of internal excitation and vibration characteristics of gears with early-stage crack and 3D tooth surface error

Affected by the tooth surface errors, the vibration characteristics of early crack failures are usually various under different tooth surface precision level. Therefore, to reveal the influence of three-dimensional (3D) tooth surface error (TSE) on the internal excitation and vibration characteristics of gears with early-stage crack (ESC), a coupling internal excitation model of gears with ESC and 3D errors is proposed based on a double-layer iterative method. In this model, the influence of meshing phase between tooth pair and along tooth width direction (TWD) on the time-varying mesh stiffness (TVMS) are simultaneously considered, which overcomes the limitations of traditional models that are only suitable for characterizing the coupled excitation of the two-dimensional tooth profile errors and TVMS. Based on verifying the proposed model, the effects of TSE type, parameters, and load on the TVMS, loaded static transmission error (LSTE), load distributions, and torsional deformation of the cracked gear teeth are investigated, and the vibration characteristics of ESC failures under the effects of 3D TSE are also explored. The research results show that the distribution parameters of 3D TSE have a significant effect on the internal excitation and vibration characteristics of gears with ESC, and thus measuring and evaluating the distribution parameters of TSE can help improve the accuracy of gear fault identification and diagnosis methods.

Isolation, Antibacterial Activity and Molecular Identification of Avocado Rhizosphere Actinobacteria as Potential Biocontrol Agents of Xanthomonas sp.

Actinobacteria, especially the genus Streptomyces, have been shown to be potential biocontrol agents for phytopathogenic bacteria. Bacteria spot disease caused by Xanthomonas spp. may severely affect chili pepper (Capsicum annuum) crops with a subsequent decrease in productivity. Therefore, the objective of the study was to isolate rhizospheric actinobacteria from soil samples treated by physical methods and evaluate the inhibitory activity of the isolates over Xanthomonas. Initially, soil samples collected from avocado tree orchards were treated by dry heat air and microwave irradiation; thereafter, isolation was implemented. Then, antibacterial activity (AA) of isolates was evaluated by the double-layer agar method. Furthermore, the positive/negative effect on AA for selected isolates was evaluated on three culture media (potato-dextrose agar, PDA; yeast malt extract agar, YME; and oat agar, OA). Isolates were identified by 16S rRNA sequence analysis. A total of 198 isolates were obtained; 76 (series BVEZ) correspond to samples treated by dry heat and 122 strains (series BVEZMW) were isolated from samples irradiated with microwaves. A total of 19 dry heat and 25 microwave-irradiated isolates showed AA with inhibition zones (IZ, diameter in mm) ranging from 12.7 to 82.3 mm and from 11.4 to 55.4 mm, respectively. An increment for the AA was registered for isolates cultured on PDA and YME, with an IZ from 21.1 to 80.2 mm and 14.1 to 69.6 mm, respectively. A lower AA was detected when isolates were cultured on OA media (15.0 to 38.1 mm). Based on the 16S rRNA gene sequencing analysis, the actinobacteria belong to the Streptomyces (6) and Amycolatopsis (2) genera. Therefore, the study showed that microwave irradiation is a suitable method to increase the isolation of soil bacteria with AA against Xanthomonas sp. In addition, Streptomyces sp. BVEZ 50 was the isolate with the highest IZ (80.2 mm).

Double-Layer Traction Method for Treating Severe Upper Auricular Adhesion Malformation in Infants: A Prospective Randomized Controlled Study.

Severe upper auricular adhesion malformation poses a significant challenge for complete correction through ear molding. This prospective randomized controlled study aimed to assess the efficacy of a new method in addressing severe upper auricular adhesions. In this non-blinded randomized controlled study, we enrolled 72 infants with severe upper auricular adhesion malformation who underwent treatment with the EarWell system. They were randomly assigned to either the double-layer traction (DLT) group or the control group. We compared the effectiveness of treatment, treatment duration, and potential complications between the two groups. All 72 infants completed the treatment and follow-up, with 38 (52.8%) in the DLT group and 34 (47.2%) in the control group. The DLT group exhibited significantly shorter treatment times and a lower incidence of pressure sores than the control group. The DLT method, along with unilateral malformation, independently predicted positive long-term outcomes. The DLT method demonstrated superior effectiveness, shorter treatment duration, and lower complication rates than traditional methods when treating severe upper auricular adhesion malformation in infants. The DLT method is safe and effective and merits broader adoption in clinical practice. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Biological and genome characteristics of a novel broad host-range phage ΦEP1 infecting enteroinvasive Escherichia coli

We analyzed the biological and genome characteristics of a phage infecting enteroinvasive Escherichia coli (EIEC), aiming to provide resources and a reference for the prevention and treatment of EIEC. With the EIEC preserved in our laboratory as the host bacterium, one strain of phage was isolated from the effluent sample from a chicken farm in Huzhou, Zhejiang and named ΦEP1. The titer, optimal multiplicity of infection, one-step growth curve, temperature, pH value, chloroform and bile salt sensitivity of ΦEP1 were determined by the double-layer agar plate method. The morphology of the phage was observed by transmission electron microscopy. The biocontrol effects of ΦEP1 in different food matrixes and the protective effect of this phage on Caco-2 cells were tested. The phage ΦEP1 showed the optimal multiplicity of infection of 0.1, the titer of 1.3×1010 PFU/mL, strong tolerance to temperature, pH, chloroform, and bile salt, and a broad host spectrum. Furthermore, it expressed lysis activity against multiple strains of multiple antibiotic-resistant pathogenic E. coli and Shigella with different serotypes. Phage ΦEP1 had an incubation period of 10 min, an outbreak period of 80 min, and an outbreak volume of 48 PFU/cell. According to the morphology observed by transmission electron microscopy, phage ΦEP1 belonged to the order of Caudovirales, and it had a good protective effect on Caco-2 cells. Phage ΦEP1 had a genome of 87 182 bp with the GC content of 39.80%, 128 putative open reading frames, and no antibiotic resistance genes or virulence genes. ΦEP1 inhibited the growth of EIEC in artificially contaminated milk and beef and eliminated EIEC in cell protection experiments. It significantly increased the survival rate of Caco-2 cells and down-regulated the expression of interleukin (IL)-6 and IL-1β to reduce inflammation. We obtained an EIEC-targeting phage ΦEP1 with a high titer and strong tolerance to the environment, which provided a basis for the application of phages in food preservation and other fields.

Screening and identification of Aspergillus sclerotiorum with activity against Metschnikowia bicuspidata and initial application on "milky disease" in Eriocheir sinensis

The "milky disease" of Chinese mitten crabs caused by Metschnikowia bicuspidata is the most serious disease of the cultivated Chinese mitten crabs in northern China, and there have been no control measures for this disease until now. In this study, a fungus 2JHP001 with a strong antagonistic effect against M. bicuspidata was screened from soil samples by the double-layer plate method and the punching method. The strain was identified as Aspergullus sclerotiorum by analyzing its morphological, molecular, physiological and biochemical characteristics. The UHPLC-MS analysis of the ethyl acetate extract showed that it contained maleic acid derivatives, terpenes, polyketides, 2,5-diketopiperazines, alkaloids, and other compounds. The morphology of yeast after treatment with 2JHP001 crude extract was observed by SEM and TEM, and the results showed that this treatment caused severe distortion of the yeast and different degrees of damage to the yeast cell walls, cell membranes and organelle. Moreover, animal experiment demonstrated that the survival rate of Chinese mitten crabs infected with "milky disease" was increased by 22.3 % within 21 d. The study provide new idea and possible strategy for the prevention and control strategy of "milky disease" in Chinese mitten crabs.

Proteomic analysis of carbapenem-resistant Klebsiella pneumoniae outer membrane vesicles under the action of phages combined with tigecycline

BackgroundKlebsiella pneumoniae is the most commonly encountered pathogen in clinical practice. Widespread use of broad-spectrum antibiotics has led to the current global dissemination of carbapenem-resistant K. pneumoniae, which poses a significant threat to antibacterial treatment efficacy and public health. Outer membrane vesicles (OMVs) have been identified as carriers capable of facilitating the transfer of virulence and resistance genes. However, the role of OMVs in carbapenem-resistant K. pneumoniae under external pressures such as antibiotic and phage treatments remains unclear.MethodsTo isolate and purify OMVs under the pressure of phages and tigecycline, we subjected K. pneumoniae 0692 harboring plasmid-mediated blaNDM-1 and blaKPC-2 genes to density gradient separation. The double-layer plate method was used to isolate MJ1, which efficiently lysed K. pneumoniae 0692 cells. Transmission electron microscopy (TEM) was used to characterize the isolated phages and extract OMV groups for relevant morphological identification. Determination of protein content of each OMV group was conducted through bicinchoninic acid assay (BCA) and proteomic analysis.ResultsK. pneumoniae 0692 released OMVs in response to different environmental stimuli, which were characterized through TEM as having the typical structure and particle size of OMVs. Phage or tigecycline treatment alone resulted in a slight increase in the mean protein concentration of OMVs secreted by K. pneumoniae 0692 compared to that in the untreated group. However, when phage treatment was combined with tigecycline, there was a significant reduction in the average protein concentration of OMVs compared to tigecycline treatment alone. Proteomics showed that OMVs encapsulated numerous functional proteins and that under different external stresses of phages and tigecycline, the proteins carried by K. pneumoniae 0692-derived OMVs were significantly upregulated or downregulated compared with those in the untreated group.ConclusionsThis study confirmed the ability of OMVs to carry abundant proteins and highlighted the important role of OMV-associated proteins in bacterial responses to phages and tigecycline, representing an important advancement in microbial resistance research.

Double-layer optimal scheduling method for solar photovoltaic thermal system based on event-triggered MPC considering battery performance degradation

Solar photovoltaic thermal system (SPTS) can fully tap solar energy resources to realize thermal and electric supply for users simultaneously, but the volatility and uncertainty of renewable energy and load cause the imbalance of energy supply. This paper proposes a multi-time scale optimal scheduling method for SPTS based on event-triggered model predictive control (ET-MPC) considering battery performance deterioration. Firstly, SPTS is divided into day-ahead and day-in optimization layers from different time scales. Day-ahead scheduling takes the minimization economic cost as the optimization function to obtain pre-scheduling plan, and day-in scheduling adapts rolling optimization scheme based on model predictive control (MPC) to reduce power fluctuations and achieve optimal scheduling adjustment. Moreover, the battery performance decay parameter is introduced into the MPC model and embedded into the optimization problem to prolong the cyclic life deterioration, and an event-triggered mechanism is introduced into MPC to improve the computational efficiency and reduce the communication frequency. Finally, the results show that the proposed double-layer scheduling method improves 4.15 %, 66 % and 13.39 % respectively regarding cost, power fluctuation and battery maintenance, and improve the calculation efficiency of 48.89 % compared with standard MPC method, which indicates that the proposed method has good economy, stability and execution efficiency.

Isolation and identification of specific Enterococcus faecalis phage C-3 and G21-7 against Avian pathogenic Escherichia coli and its application to one-day-old geese.

Colibacillosis caused by Avian pathogenic Escherichia coli (APEC), including peritonitis, respiratory tract inflammation and ovaritis, is recognized as one of the most common and economically destructive bacterial diseases in poultry worldwide. In this study, the characteristics and inhibitory potential of phages were investigated by double-layer plate method, transmission electron microscopy, whole genome sequencing, bioinformatics analysis and animal experiments. The results showed that phages C-3 and G21-7 isolated from sewage around goose farms infected multiple O serogroups (O1, O2, O18, O78, O157, O26, O145, O178, O103 and O104) Escherichia coli (E.coli) with a multiplicity of infection (MOI) of 10 and 1, respectively. According to the one-step growth curve, the incubation time of both bacteriophage C-3 and G21-7 was 10 min. Sensitivity tests confirmed that C-3 and G21-6 are stable at 4 to 50 °C and pH in the range of 4 to 11. Based on morphological and phylogenetic analysis, phages C-3 and G21-7 belong to Enterococcus faecalis (E. faecalis) phage species of the genus Saphexavirus of Herelleviridae family. According to genomic analysis, phage C-3 and G21-7 were 58,097 bp and 57,339 bp in size, respectively, with G+C content of 39.91% and 39.99%, encoding proteins of 97 CDS (105 to 3,993 bp) and 96 CDS (105 to 3,993 bp), and both contained 2 tRNAs. Both phages contained two tail proteins and holin-endolysin system coding genes, and neither carried resistance genes nor virulence factors. Phage mixture has a good safety profile and has shown good survival probability and feed efficiency in both treatment and prophylaxis experiments with one-day-old goslings. These results suggest that phage C-3 and G21-7 can be used as potential antimicrobials for the prevention and treatment of APEC.

Integrated control strategy for 5G base station frequency regulation considering spatio-temporal variations in communication load

The decreasing system inertia and active power reserves caused by the penetration of renewable energy sources and the displacement of conventional generating units present new challenges to the frequency stability of modern power systems. Vast quantities of 5G base stations, featuring largely dormant battery storage systems and advanced communication technology, represent a high-quality fast frequency regulation resource for the power system. This paper proposes a double-layer clustering method for 5G base stations and an integrated centralized-decentralized control strategy for their participation in frequency regulation, considering the variability of available frequency regulation capacity due to spatial and temporal variations in base station communication loads. The proposed capacity model and control methods are evaluated using a case study of a two-machine test system with 10,000 real 5G base stations, demonstrating the effectiveness of the clustering method and the integrated control strategy in improving frequency stability.

Evaluation of the application potential of Bdellovibrio sp. YBD-1 isolated from Yak faeces

Studies on Bdellovibrio and like organisms (BALOs), obligate predatory bacteria, have highlighted the possibility of regulating bacteria and biofilms; however, yak-derived BALOs are yet to be reported. We aimed to characterize the BALOs isolated and identified from yak (Bos grunniens) feces and examine application potential. BALOs were isolated from healthy yak fecal samples, with Escherichia coli (ATCC 25922) as prey using the double-layer agar method, identified by transmission electron microscopy (TEM), and the specific 16S rDNA sequencing analysis. Sequencing of the 16S rDNA gene indicated that this isolate was 91% similar to the Bdellovibrio sp. NC01 reference strain and was named YBD-1. Proportion of YBD-1 lysed bacteria is 12/13. The YBD-1 showed best growth at 25–40°C, 0–0.25% (w/v) NaCl, and pH 6.5–7.5. YBD-1 significantly reduced the planktonic cells and biofilms of E.coli in co-culture compared to the E.coli group. Additionally, SEM analysis indicated that YBD-1 significantly reduced biofilm formation in E. coli. Furthermore, quantitative Real Time-polymerase chain reaction (qRT-PCR) showed that the expression of the virulence genes fim and iroN and the genes pgaABC involved in biofilm formation went down significantly. We concluded that YBD-1 may have the potential to control bacterial growth and biofilm-associated bacterial illnesses.

Precision Engineering of the Co-immobilization of Enzymes for Cascade Biocatalysis.

The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N-terminus of an alcohol dehydrogenase (ADH) and an aldo-keto reductase (AKR), respectively. A non-canonical amino acid (ncAA), p-azido-L-phenylalanine (p-AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide-alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual-enzyme coating on porous microspheres. The ordered dual-enzyme reactor was subsequently used to synthesize (S)-1-(2-chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double-layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single-layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

Biological characterization and complete genome analysis of the newly isolated Serratia liquefaciens phage vB_SlqS_ZDD2.

A novel lytic phage named vB_SlqS_ZDD2 was isolated from hospital sewage using the double-layer agar method with Serratia liquefaciens ATCC 27592 as the host. BLASTn analysis showed that the genome sequence of phage vB_SlqS_ZDD2 did not resemble any other phages in the NCBI database. Phenotype and phylogeny analysis indicated that this phage might be a new member of the class Caudoviricetes. Phage vB_SlqS_ZDD2 has a dsDNA genome of 49,178 bp with 55% GC content and has 73 open reading frames. This phage exhibited strong lytic activity and a wide range of pH (3-12) and temperature tolerance (below 70℃).

Characterization and resistance mechanism of phage-resistant strains of Salmonella enteritidis

In the face of the increasingly severe problem of antibiotic resistance, phage therapy is regarded as a highly potential alternative. Compared with traditional antimicrobial agents, a key research area of phage therapy is the study of phage-resistant mutant bacteria. To effectively monitor and prevent this resistance, it is crucial to conduct in-depth exploration of the mechanism behind phage resistance. In this study, a strain of Salmonella enteritidis (sm140) and the corresponding phage (Psm140) were isolated from chicken liver and sewage, respectively. Using the double-layer plate method, successfully screened out phage-resistant mutant strains. Whole-genome resequencing of 3 resistant strains found that the wbaP gene of all 3 strains had mutations at a specific position (1,118), with the base changing from G to A. This mutation causes the gene-encoded glycine to be replaced by aspartic acid. Subsequent studies found that the frequency of this gene mutation is extremely high, reaching 84%, and all mutations occur at the same position. To further explore the relationship between the wbaP gene and phage resistance, knockout strains and complement strains of the wbaP gene were constructed. The experimental results confirmed the association between the wbaP gene and phage resistance. At the same time, biological characteristics and virulence were evaluated for wild strains, resistant strains, knockout strains, and complement strains. It was found that mutations or deletions of the wbaP gene lead to a decrease in bacterial environmental adaptability and virulence. Through systematic research on the mechanism and biological characteristics of phage resistance, this study provides important references and guidance for the development of new phage therapies, promoting progress in the field of antimicrobial treatment. At the same time, the emergence of phage resistance due to wbaP gene mutations is reported for the first time in salmonella, providing a new perspective and ideas for further studying phage resistance mechanisms.

Immersed Boundary Double Layer method: An introduction of methodology on the Helmholtz equation

The Immersed Boundary (IB) method of Peskin (1977) [1] is useful for problems that involve fluid-structure interactions or complex geometries. By making use of a regular Cartesian grid that is independent of the geometry, the IB framework yields a robust numerical scheme that can efficiently handle immersed deformable structures. Additionally, the IB method has been adapted to problems with prescribed motion and other PDEs with given boundary data. IB methods for these problems traditionally involve penalty forces which only approximately satisfy boundary conditions, or they are formulated as constraint problems. In the latter approach, one must find the unknown forces by solving an equation that corresponds to a poorly conditioned first-kind integral equation. This operation can therefore require a large number of iterations of a Krylov method, and since a time-dependent problem requires this solve at each step in time, this method can be prohibitively inefficient without preconditioning. This work introduces a new, well-conditioned IB formulation for boundary value problems, called the Immersed Boundary Double Layer (IBDL) method. In order to lay the groundwork for similar formulations of Stokes and Navier-Stokes equations, this paper focuses on the Poisson and Helmholtz equations to introduce the methodology and to demonstrate its efficiency over the original constraint method. In this double layer formulation, the equation for the unknown boundary distribution corresponds to a well-conditioned second-kind integral equation that can be solved efficiently with a small number of iterations of a Krylov method without preconditioning. Furthermore, the iteration count is independent of both the mesh size and spacing of the immersed boundary points. The method converges away from the boundary, and when combined with a local interpolation, it converges in the entire PDE domain. Additionally, while the original constraint method applies only to Dirichlet problems, the IBDL formulation can also be used for Neumann boundary conditions.

Application research on low cost support method for surrounding rock of broken section roadway in phosphate mine area

Introduction: Taking a phosphate mine in Yunnan as the engineering research background, the fractured rock mass of the No. 1 belt roadway is selected as the research object.Methods: To reduce the cost of tunnel surrounding rock support and optimize construction technology, a combination of numerical simulation software Midas and on-site experiments was used to compare and analyze the distribution characteristics of the plastic zone of tunnel surrounding rock, deformation characteristics of tunnel surrounding rock, and stress characteristics of tunnel support anchor bolts under two support methods: double-layer spray anchor mesh and single-layer spray anchor mesh with steel bar.Results: Research findings indicate that adopting the single-layer spray anchor mesh with steel bar support method results in a more uniform distribution of the plastic zone at the top of the tunnel surrounding rock, with a smaller range. The settlement of the roof and bulging of the bottom plate in the roadway’s surrounding rock, supported by a single-layer spray anchor mesh and steel bars, measure at 75.033 mm and 82.165 mm, respectively. Additionally, the internal bulging on both sides of the roadway’s surrounding rock is recorded as 37.835 mm and 37.470 mm, respectively. On the other hand, when utilizing a double-layer spray anchor mesh for support, the settlement of the roof and bulging of the bottom plate in the roadway’s surrounding rock are measured at 84.199 mm and 85.255 mm, respectively. Correspondingly, the internal bulging on both sides of the roadway’s surrounding rock records measurements of 41.670 mm and 41.756 mm, respectively. Furthermore, adopting the single-layer spray anchor mesh with steel bar support method ensures a more even transfer of axial forces to the steel bars, thereby increasing the overall load-bearing capacity. In contrast, the anchor bolts in the double-layer spray anchor mesh support method experience higher axial forces, leading to more pronounced stress concentration.Discussion: By comparing the field monitoring data, it is evident that the single-layer spray anchor mesh with steel bar support method exhibits significantly better control over the tunnel surrounding rock than the double-layer spray anchor mesh support method. Therefore, replacing the double-layer spray anchor net support with a single-layer spray anchor net with steel bar support method is entirely feasible and can meet on-site practical production requirements. The research results hold significant value in providing guidance and reference for selecting cost-effective support schemes for tunnel surrounding rock.

Endophytic Stenotrophomonas geniculata KJ-6 via producing antifungal volatile organic compounds effectively control Lanzhou lily postharvest diseases

The endophytic bacterial Stenotrophomonas geniculata KJ-6, was isolated from Arisaematis rhizoma. This strain has the ability to control three postharvest pathogenic fungi of Lanzhou lily (Trichoderma lixii F-2, Talaromyces tumuli F-3, and Fusarium annulatum F-6) by releasing volatile organic compounds (VOCs). The experiment utilized the double-layer sealed bottom plate method. The results demonstrate that KJ-6 effectively inhibits the mycelial growth of F-2, F-3, and F-6, with inhibition rates of 81.59%, 79.87%, and 84.7%, respectively. Moreover, scanning electron microscopy (SEM) revealed abnormal mycelium morphology in the test pathogenic fungi treated with KJ-6 VOCs, characterized by multiple wrinkles, cracks, and distortions. Using five common phytopathogenic fungi as controls, our results demonstrated that the VOCs from KJ-6 exhibited broad-spectrum antifungal effects. Analysis using headspace gas chromatography-ion mobility spectrometry identified three components of the VOCs produced by KJ-6, including 1-propanethiol, isoamyl acetate, and 2-methyl-1-butanol. Antifungal tests revealed that 1-propanethiol plays a crucial role in the inhibitory activity of VOCs. It exhibited inhibitory rates of 64.17%, 72.73%, and 26.71% on the mycelium growth of F-2, F-3, and F-6, respectively. In vivo experiments further confirmed that VOCs can significantly reduce the disease index and decay rate of Lanzhou lily bulbs during storage. Moreover, KJ-6 treatment led to a significant reduction in the degree of browning of lily bulbs. Additionally, it enhanced the activity of defense-related enzymes, improved disease resistance, and extended the shelf life of lily bulbs. As a result, this study offers a theoretical foundation for the biological control of postharvest diseases in Lanzhou lily.