Rapid Identification of Brucella Genus and Species In Silico and On-Site Using Novel Probes with CRISPR/Cas12a.

Human brucellosis caused by Brucella is a widespread zoonosis that is prevalent in many countries globally. The high homology between members of the Brucella genus and Ochrobactrum spp. often complicates the determination of disease etiology in patients. The efficient and reliable identification and distinction of Brucella are of primary interest for both medical surveillance and outbreak purposes. A large amount of genomic data for the Brucella genus was analyzed to uncover novel probes containing single-nucleotide polymorphisms (SNPs). GAMOSCE v1.0 software was developed based on the above novel eProbes. In conjunction with clinical requirements, an RPA-Cas12a detection method was developed for the on-site determination of B. abortus and B. melitensis by fluorescence and lateral flow dipsticks (LFDs). We demonstrated the potential of these probes for rapid and accurate detection of the Brucella genus and five significant Brucella species in silico using GAMOSCE. GAMOSCE was validated on different Brucella datasets and correctly identified all Brucella strains, demonstrating a strong discrimination ability. The RPA-Cas12a detection method showed good performance in detection in clinical blood samples and veterinary isolates. We provide both in silico and on-site methods that are convenient and reliable for use in local hospitals and public health programs for the detection of brucellosis.

Engineered Microrobots for Targeted Delivery of Bacterial Outer Membrane Vesicles (OMV) in Thrombus Therapy.

In the realm of thrombosis treatment, bioengineered outer membrane vesicles (OMVs) offer a novel and promising approach, as they have rich content of bacterial-derived components. This study centers on OMVs derived from Escherichia coli BL21 cells, innovatively engineered to encapsulate the staphylokinase-hirudin fusion protein (SFH). SFH synergizes the properties of staphylokinase (SAK) and hirudin (HV) to enhance thrombolytic efficiency while reducing the risks associated with re-embolization and bleeding. Building on this foundation, this study introduces two cutting-edge microrobotic platforms: SFH-OMV@H for venous thromboembolism (VTE) treatment, and SFH-OMV@MΦ, designed specifically for cerebral venous sinus thrombosis (CVST) therapy. These platforms have demonstrated significant efficacy in dissolving thrombi, with SFH-OMV@H showcasing precise vascular navigation and SFH-OMV@MΦ effectively targeting cerebral thrombi. The study shows that the integration of these bioengineered OMVs and microrobotic systems marks a significant advancement in thrombosis treatment, underlining their potential to revolutionize personalized medical approaches to complex health conditions.

Aging-related biomarker discovery in the era of immune checkpoint inhibitors for cancer patients.

Older patients with cancer, particularly those over 75 years of age, often experience poorer clinical outcomes compared to younger patients. This can be attributed to age-related comorbidities, weakened immune function, and reduced tolerance to treatment-related adverse effects. In the immune checkpoint inhibitors (ICI) era, age has emerged as an influential factor impacting the discovery of predictive biomarkers for ICI treatment. These age-linked changes in the immune system can influence the composition and functionality of tumor-infiltrating immune cells (TIICs) that play a crucial role in the cancer response. Older patients may have lower levels of TIICs infiltration due to age-related immune senescence particularly T cell function, which can limit the effectivity of cancer immunotherapies. Furthermore, age-related immune dysregulation increases the exhaustion of immune cells, characterized by the dysregulation of ICI-related biomarkers and a dampened response to ICI. Our review aims to provide a comprehensive understanding of the mechanisms that contribute to the impact of age on ICI-related biomarkers and ICI response. Understanding these mechanisms will facilitate the development of treatment approaches tailored to elderly individuals with cancer.

Steering Electron-Induced Surface Reaction via a Molecular Assembly Approach.

Electrons not only serve as a "reactant" in redox reactions but also play a role in "catalyzing" some chemical processes. Despite the significance and ubiquitousness of electron-induced chemistry, many related scientific issues still await further exploration, among which is the impact of molecular assembly. In this work, microscopic insights into the vital role of molecular assembly in tweaking the electron-induced surface chemistry are unfolded by combined scanning tunneling microscopy and density functional theory studies. It is shown that the selective dissociation of a C-Cl bond in 4,4″-dichloro-1,1':3',1''-terphenyl (DCTP) on Cu(111) can be efficiently triggered by an electron injection via the STM tip into the unoccupied molecular orbital. The DCTP molecules are embedded in different assembly structures, including its self-assembly and coassemblies with Br adatoms. The energy threshold for the C-Cl bond cleavage increases as more Br adatoms stay close to the molecule, indicative of the sensitive response of the electron-induced surface reactivity of the C-Cl bond to the subtle change in the molecular assembly. Such a phenomenon is rationalized by the energy shift of the involved unoccupied molecular orbital of DCTP that is embedded in different assemblies. These findings shed new light on the tuning effect of molecular assembly on electron-induced reactions and introduce an efficient approach to precisely steer surface chemistry.

Relationship between autoimmune thyroid antibodies and anti-nuclear antibodies in general patients.

Background: There is no doubt that both Hashimoto thyroiditis and Graves' disease are autoimmune thyroid diseases (AITDs), but the relationship between anti-nuclear antibody (ANA) and AITDs is poorly studied. The association between thyroid autoantibody levels and ANA positivity was evaluated to assess the role of ANA in AITDs. Methods: We conducted an analysis using data from 1,149,893 patients registered at our hospital and 53,021 patients registered in the National Health and Nutrition Examination Survey databases. We focused on patients with data for thyroid peroxidase antibody (TPOAb)/ANA, TPOAb/immunoglobulin G (IgG), thyroid-stimulating hormone (TSH) receptor antibody (TRAb)/ANA, TRAb/IgG, TSH/ANA, or TSH/IgG. Results: ANA positivity rates were 12.88% and 21.22% in TPOAb/ANA and TSH/ANA patients, respectively. In TPOAb/IgG and TSH/IgG data, high IgG levels (≥15 g/L) were detected in 2.23% and 4.06% of patients, respectively. There were significant differences in ANA positivity rates and high IgG proportions among patients with different TPOAb and TSH levels. TPOAb level was correlated with ANA positivity rate and high IgG proportion, and TSH level was correlated with ANA positivity rate. Regression analysis showed positive correlations between TPOAb levels and ANA positivity risk or high IgG risk, TSH levels and high IgG risk, and elevated TSH and ANA positivity risk. Of patients with TRAb/ANA data, 35.99% were ANA-positive, and 13.93% had TRAb levels ≥1.75IU/L; 18.96% of patients with TRAb/IgG data had high IgG levels, and 16.51% had TRAb levels ≥1.75IU/L. ANA positivity rate and high IgG proportion were not significantly different among different TRAb levels. TRAb levels, ANA positivity risk and high IgG risk were not correlated. Conclusion: ANA positivity and high IgG are related to Hashimoto thyroiditis but not Graves' disease, which implies distinct pathophysiological mechanisms underlying the AITDs.

Analysis of carbapenem-resistant Acinetobacter baumannii carbapenemase gene distribution and biofilm formation.

OBJECTIVE: In recent years, Acinetobacter baumannii has been appearing in hospitals with high drug resistance and strong vitality, which brings many difficulties to clinical treatment. In this study, 255 strains of A. baumannii were isolated from Youjiang Medical University for Nationalities Affiliated Hospital clinical samples and found to be highly resistant to carbapenems. The drug resistance, biofilm-forming ability, and carbapenase gene distribution of 145 carbapenem-resistant A. baumannii (CRAB) strains were analyzed statistically. METHODS: The clinically isolated strains were detected using Vitek mass spectrometry and Vitek2-compact for bacterial identification and susceptibility testing, respectively. The biofilms of clinical isolates were quantitatively detected by microplate crystal violet staining, and qualitatively observed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). And the common carbapenemase genes were detected by polymerase chain reaction (PCR). RESULTS: The 255 clinical isolates from the Youjiang District of western Guangxi Province had a high resistance rate to carbapenems antibiotics. The main specimens were from the intensive care unit (49%), and the most important specimens were sputum specimens (80%). All 145 strains of CRAB produced different degrees of biofilm, and six carbapenenase genes were detected. We found that there were significant differences in biofilm formation between resistant and sensitive strains of tobramycin, levofloxacin, ciprofloxacin, tigecycline, and doxycycline (P<0.05). The distribution of blaOXA-23 and blaOXA51 genes was significantly different from CRAB biofilm formation (P<0.05). In addition, AmpC, blaOXA-23, blaOXA-51, and TEM genes were more distributed in antibiotic-resistant strains. CONCLUSION: The clinical strains have a high resistance rate to carbapenems, and the CRAB with blaOXA-51 and blaOXA-23 genes has a high resistance to antibiotics and a strong biofilm.

DR6 Augments Colorectal Cancer Cell Growth, Invasion, and Stemness by Activating AKT/NF-κB Pathway.

This study aims to elucidate the role and mechanisms of Death Receptor 6 (DR6), a member of the tumor necrosis factor receptor superfamily, in the malignant progression of colorectal cancer (CRC). The association of DR6 expression levels and CRC patient survival was examined using the CRC cohort data from GEPIA database. The functional role of DR6 in CRC cells was investigated by performing loss-of-function and gain-of-function experiments based on CCK-8 proliferation assay, transwell migration and invasion assay, and sphere-forming assays. Xenograft model of CRC cells in nude mouse was established to evaluate the impact of DR6 knockdown on CRC tumorigenesis. Elevated expression of DR6 was correlated with an unfavorable prognosis in CRC patients. In vitro functional assays demonstrated that silencing DR6 considerably suppressed the proliferation, migration, invasion, and stemness of CRC cells, whereas its overexpression showed an opposite effect. DR6 knockdown also attenuated tumor formation of CRC cells in the nude mice. Mechanistically, silencing DR6 reduced the phosphorylation of AKT and NF-κB in CRC cells, and the treatment with an AKT activator (SC79) abrogated the inhibitory effects of DR6 knockdown on the malignant features of CRC cells. Our data suggest that DR6 contributes to the malignant progression of CRC by activating AKT/NF-κB pathway, indicating its clinical potential as a prognostic marker and therapeutic target for CRC.

A Serious Game ("Fight With Virus") for Preventing COVID-19 Health Rumors: Development and Experimental Study.

BACKGROUND: Health rumors arbitrarily spread in mainstream social media on the internet. Health rumors emerged in China during the outbreak of COVID-19 in early 2020. Many midelders/elders (age over 40 years) who lived in Wuhan believed these rumors. OBJECTIVE: This study focused on designing a serious game as an experimental program to prevent and control health rumors. The focus of the study was explicitly on the context of the social networking service for midelders/elders. METHODS: This research involved 2 major parts: adopting the Transmission Control Protocol model for games and then, based on the model, designing a game named "Fight With Virus" as an experimental platform and developing a cognitive questionnaire with a 5-point Likert scale. The relevant variables for this experimental study were defined, and 10 hypotheses were proposed and tested with an empirical study. In total, 200 participants were selected for the experiments. By collecting relevant data in the experiments, we conducted statistical observations and comparative analysis to test whether the experimental hypotheses could be proved. RESULTS: We noted that compared to traditional media, serious games are more capable of inspiring interest in research participants toward their understanding of the knowledge and learning of health commonsense. In judging and recognizing the COVID-19 health rumor, the test group that used game education had a stronger ability regarding identification of the rumor and a higher accuracy rate of identification. Results showed that the more educated midelders/elders are, the more effective they are at using serious games. CONCLUSIONS: Compared to traditional media, serious games can effectively improve midelders'/elders' cognitive abilities while they face a health rumor. The gameplay effect is related to the individual's age and educational background, while income and gender have no impact.

Biomechanical evaluation on a new type of vertebral titanium porous mini-plate and mechanical comparison between cervical open-door laminoplasty and laminectomy: a finite element analysis.

Objective: Compare the spine's stability after laminectomy (LN) and laminoplasty (LP) for two posterior surgeries. Simultaneously, design a new vertebral titanium porous mini plate (TPMP) to achieve firm fixation of the open-door vertebral LP fully. The objective is to enhance the fixation stability, effectively prevent the possibility of "re-closure," and may facilitate bone healing. Methods: TPMP was designed by incorporating a fusion body and porous structures, and a three-dimensional finite element cervical model of C2-T1 was constructed and validated. Load LN and LP finite element models, respectively, and analyze and simulate the detailed processes of the two surgeries. It was simultaneously implanting the TPMP into LP to evaluate its biomechanical properties. Results: We find that the range of motion (ROM) of C4-C5 after LN surgery was greater than that of LP implanted with different plates alone. Furthermore, flexion-extension, lateral bending, and axial rotation reflect this change. More noteworthy is that LN has a much larger ROM on C2-C3 in axial rotation. The ROM of LP implanted with two different plates is similar. There is almost no difference in facet joint stress in lateral bending. The facet joint stress of LN is smaller on C2-C3 and C4-C5, and larger more prominent on C5-C6 in the flexion-extension. Regarding intervertebral disc pressure (IDP), there is little difference between different surgeries except for the LN on C2-C3 in axial rotation. The plate displacement specificity does not significantly differ from LP with vertebral titanium mini-plate (TMP) and LP with TPMP after surgery. The stress of LP with TPMP is larger in C4-C5, C5-C6. Moreover, LP with TMP shows greater stress in the C3-C4 during flexion-extension and lateral bending. Conclusion: LP may have better postoperative stability when posterior approach surgery is used to treat CSM; at the same time, the new type of vertebral titanium mini-plate can achieve almost the same effect as the traditional titanium mini-plate after surgery for LP. In addition, it has specific potential due to the porous structure promoting bone fusion.

Surface-confined alternating copolymerization with molecular precision by stoichiometric control.

Keen desires for artificial mimicry of biological polymers and property improvement of synthesized ones have triggered intensive explorations for sequence-controlled copolymerization. However, conventional synthesis faces great challenges to achieve this goal due to the strict requirements on reaction kinetics of comonomer pairs and tedious synthetic processes. Here, sequence-controlled alternating copolymerization with molecular precision is realized on surface. The stoichiometric control serves as a thermodynamic strategy to steer the polymerization selectivity, which enables the selective alternating organometallic copolymerization via intermolecular metalation of 4,4"-dibromo-p-terphenyl (P-Br) and 2,5-diethynyl-1,4-bis(phenylethynyl)benzene (A-H) with Ag adatoms on Ag(111) at P-Br: A-H = 2, as verified by scanning tunneling microscopy and density functional theory studies. In contrast, homopolymerization yield increases as the stoichiometric ratio deviates from 2. The microscopic characterizations rationalize the mechanism, providing a delicate explanation of the stoichiometry-dependent polymerization. These findings pave a way to actualizing an efficient sequence control of copolymerization by surface chemistry.

Substituent Positioning Effects on the Magnetic Properties of Sandwich-Type Erbium(III) Complexes with Bis(trimethylsilyl)-Substituted Cyclooctatetraenyl Ligands.

Lanthanide complexes with judiciously designed ligands have been extensively studied for their potential applications as single-molecule magnets. With the influence of ligands on their magnetic properties generally established, recent research has unearthed certain effects inherent to site differentiation due to the different types and varying numbers of substituents on the same ligand platform. Using two new sandwich-type Er(III) complexes with cyclooctatetraenyl (COT) ligands featuring two differently positioned trimethylsilyl (TMS) substituents, namely, [Li(DME)Er(COT1,5-TMS2)2]n (Er1) and [Na(DME)3][Er(COT1,3-TMS2)2] (Er2) [COT1,3-TMS2 and COT1,5-TMS2 donate 1,3- and 1,5-bis(trimethylsilyl)-substituted cyclooctatetraenyl ligands, respectively; DME = 1,2-dimethoxyethane], and with reference to previously reported [Li(DME)3][Er(COT1,4-TMS2)2] (A) and [K(DME)2][Er(COT1,4-TMS2)2] (B), any possible substituent position effects have been explored for the first time. The rearrangement of the TMS substituents from the starting COT1,4-TMS2 to COT1,3-TMS2 and COT1,5-TMS2, by way of formal migration of the TMS group, was thermally induced in the case of Er1, while for the formation of Er2, the use of Na+ in the placement of its Li+ and K+ congeners is essential. Both Er1 and Er2 display single-molecule magnetic behaviors with energy barriers of 170(3) and 172(6) K, respectively. Magnetic hysteresis loops, butterfly-shaped for Er1 and wide open for Er2, were observed up to 12 K for Er1 and 13 K for Er2. Studies of magnetic dynamics reveal the different pathways for relaxation of magnetization below 10 K, mainly by the Raman process for Er1 and by quantum tunneling of magnetization for Er2, leading to the order of magnitude difference in magnetic relaxation times and sharply different magnetic hysteresis loops.

Individualized treatment decision model for inoperable elderly esophageal squamous cell carcinoma based on multi-modal data fusion.

BACKGROUND: This research aimed to develop a model for individualized treatment decision-making in inoperable elderly patients with esophageal squamous cell carcinoma (ESCC) using machine learning methods and multi-modal data. METHODS: A total of 189 inoperable elderly ESCC patients aged 65 or older who underwent concurrent chemoradiotherapy (CCRT) or radiotherapy (RT) were included. Multi-task learning models were created using machine learning techniques to analyze multi-modal data, including pre-treatment CT images, clinical information, and blood test results. Nomograms were constructed to predict the objective response rate (ORR) and progression-free survival (PFS) for different treatment strategies. Optimal treatment plans were recommended based on the nomograms. Patients were stratified into high-risk and low-risk groups using the nomograms, and survival analysis was performed using Kaplan-Meier curves. RESULTS: The identified risk factors influencing ORR were histologic grade (HG), T stage and three radiomic features including original shape elongation, first-order skewness and original shape flatness, while risk factors influencing PFS included BMI, HG and three radiomic features including high gray-level run emphasis, first-order minimum and first-order skewness. These risk factors were incorporated into the nomograms as independent predictive factors. PFS was substantially different between the low-risk group (total score ≤ 110) and the high-risk group (total score > 110) according to Kaplan-Meier curves (P < 0.05). CONCLUSIONS: The developed predictive models for ORR and PFS in inoperable elderly ESCC patients provide valuable insights for predicting treatment efficacy and prognosis. The nomograms enable personalized treatment decision-making and can guide optimal treatment plans for inoperable elderly ESCC patients.

Effect of adjuvant radiotherapy after breast-conserving surgery in elder women with early-stage breast cancer: a propensity-score matching analysis.

Purpose: The study aimed to explore the role of adjuvant radiotherapy (RT) after breast-conserving surgery (BCS) in elder women with early-stage breast cancer (BC). Methods: BC patients with 70-79 years of age, stage T1-2N0-1M0, undergoing BCS were screened in the Surveillance, Epidemiology and End Results (SEER) database between 2010 and 2015. The clinicopathological characteristics were balanced with propensity-score matching (PSM) method. Kaplan-Meier curves and Cox regression analyses were performed to determine the impact of adjuvant RT on BC patients. Results: Ultimately, 12,310 patients treated with adjuvant RT and 4837 patients treated with no RT, were involved in the analysis. Overall, patients treated with adjuvant RT was associated with a better breast cancer-specific survival (BCSS) (HR: 1.980 [1.596- 2.456], P < 0.001) and overall survival (OS) (HR: 2.214 [1.966- 2.494], P < 0.001) than those who did not undergo RT. After 1:1 PSM, adjuvant RT still performed advantage in both BCSS (HR: 1.918 [1.439- 2.557], P < 0.001) and OS (HR: 2.235 [1.904- 2.624], P < 0.001). In the multivariate COX analysis of BCSS, widowed, divorced and separated patients, tumor grade III, T2 stage, N1 stage, no RT, molecular subtypes with luminal B and triple negative were associated with a shorter BCSS (P < 0.05). In the multivariate COX analysis of OS, age ≥74 years, widowed, divorced and separated patients, tumor grade II/III, T2 stage, no RT, no chemotherapy, molecular subtypes with triple negative were associated with a shorter OS (P < 0.05). Furthermore, the advantages of adjuvant RT were observed in all subgroup analysis. Conclusion: Adjuvant RT after BCS can improve both BCSS and OS in elderly patients with early-stage BC. Additionally, all subgroups analysis-derived BCSS and OS were in support of RT.

Value Added Conversion of Ethanol on Morphologically Controlled and Defect-Engineered Titanium Dioxide Nanorods.

Developing an environmentally benign and highly effective strategy for the value-added conversion of biomass platform molecules such as ethanol has emerged as a significant challenge and opportunity. This challenge stems from the need to harness renewable solar energy and conduct thermodynamically unfavorable reactions at room temperature. To tackle this challenge, one-dimensional titanium dioxide photocatalysts have been designed and fabricated to achieve a remarkable photocatalytic selectivity of almost 100 % for transforming ethanol into value-added 1,1-diethoxyethane, contrasting the primary production of acetaldehyde in titanium dioxide nanoparticles. By incorporating a Pt co-catalyst and infusing oxygen vacancies into the one-dimensional catalyst, the ethanol transformation rate was doubled to 128.8 mmol g-1 h-1 with respect to that of its unmodified counterpart (about 66.7 mmol g-1 h-1 ). The underlying mechanism for this high conversion and selectivity resides in the narrowed bandgap of the catalyst and the prolonged lifetime of the photo-generated carriers. This is a promising strategy for the photocatalytic transformation of essential biomass platform molecules that intertwines morphological control and defect engineering.

Clustering-Evolved Frontier Orbital for Low-Temperature CO2 Dissociation.

In this study, single Ni2 clusters (two Ni atoms bridged by a lattice oxygen) are successfully synthesized on monolayered CuO. They exhibit a remarkable activity toward low-temperature CO2 thermal dissociation, in contrast to cationic Ni atoms that nondissociatively adsorb CO2 and metallic Ni ones that are chemically inert for CO2 adsorption. Density functional theory calculations reveal that the Ni2 clusters can significantly alter the spatial symmetry of their unoccupied frontier orbitals to match the occupied counterpart of the CO2 molecule and enable its low-temperature dissociation. This study may help advance single-cluster catalysis and exploit the unexcavated mechanism for low-temperature CO2 activation.

Autonomous metal-organic framework nanorobots for active mitochondria-targeted cancer therapy.

Nanorobotic manipulation to access subcellular organelles remains unmet due to the challenge in achieving intracellular controlled propulsion. Intracellular organelles, such as mitochondria, are an emerging therapeutic target with selective targeting and curative efficacy. We report an autonomous nanorobot capable of active mitochondria-targeted drug delivery, prepared by facilely encapsulating mitochondriotropic doxorubicin-triphenylphosphonium (DOX-TPP) inside zeolitic imidazolate framework-67 (ZIF-67) nanoparticles. The catalytic ZIF-67 body can decompose bioavailable hydrogen peroxide overexpressed inside tumor cells to generate effective intracellular mitochondriotropic movement in the presence of TPP cation. This nanorobot-enhanced targeted drug delivery induces mitochondria-mediated apoptosis and mitochondrial dysregulation to improve the in vitro anticancer effect and suppression of cancer cell metastasis, further verified by in vivo evaluations in the subcutaneous tumor model and orthotopic breast tumor model. This nanorobot unlocks a fresh field of nanorobot operation with intracellular organelle access, thereby introducing the next generation of robotic medical devices with organelle-level resolution for precision therapy.

Isolation and Characterization of Three Pseudomonas aeruginosa Viruses with Therapeutic Potential.

As one of the most common pathogens of opportunistic and hospital-acquired infections, Pseudomonas aeruginosa is associated with resistance to diverse antibiotics, which represents a significant challenge to current treatment modalities. Phage therapy is considered a promising alternative to conventional antimicrobials. The characterization and isolation of new bacteriophages and the concurrent evaluation of their therapeutic potential are fundamental for phage therapy. In this study, we employed an enrichment method and a double-layer agar overlay to isolate bacteriophages that infect P. aeruginosa strains PAO1 and PA14. Three phages (named PA_LZ01, PA_LZ02, and PA_LZ03) were isolated and showed icosahedral heads and contractile tails. Following full-genome sequencing, we found that phage PA_LZ01 contained a genome of 65,367 bp in size and harbored 90 predicted open reading frames (ORFs), phage PA_LZ02 contained a genome of 57,243 bp in size and harbored 75 predicted ORFs, and phage PA_LZ03 contained a genome of 57,367 bp in size and carried 77 predicted ORFs. Further comparative analysis showed that phage PA_LZ01 belonged to the genus Pbunavirus genus, phage PA_LZ02 belonged to the genus Pamexvirus, and phage PA_LZ03 belonged to the family Mesyanzhinovviridae. Next, we demonstrated that these phages were rather stable at different temperatures and pHs. One-step growth curves showed that the burst size of PA_LZ01 was 15 PFU/infected cell, and that of PA_LZ02 was 50 PFU/infected cell, while the titer of PA_LZ03 was not elevated. Similarly, the biofilm clearance capacities of PA_LZ01 and PA_LZ02 were also higher than that of PA_LZ03. Therapeutically, PA_LZ01 and PA_LZ02 treatment led to decreased bacterial loads and inflammatory responses in a mouse model. In conclusion, we isolated three phages that can infect P. aeruginosa, which were stable in different environments and could reduce bacterial biofilms, suggesting their potential as promising candidates to treat P. aeruginosa infections. IMPORTANCE Phage therapy is a promising therapeutic option for treating bacterial infections that do not respond to common antimicrobial treatments. Biofilm-mediated infections are particularly difficult to treat with traditional antibiotics, and the emergence of antibiotic-resistant strains has further complicated the situation. Pseudomonas aeruginosa is a bacterial pathogen that causes chronic infections and is highly resistant to many antibiotics. The library of phages that target P. aeruginosa is expanding, and the isolation of new bacteriophages is constantly required. In this study, three bacteriophages that could infect P. aeruginosa were isolated, and their biological characteristics were investigated. In particular, the isolated phages are capable of reducing biofilms formed by P. aeruginosa. Further analysis indicates that treatment with PA_LZ01 and PA_LZ02 phages reduces bacterial loads and inflammatory responses in vivo. This study isolated and characterized bacteriophages that could infect P. aeruginosa, which offers a resource for phage therapy.

EptA of Riemerella anatipestifer mediates phenotypes involved in colistin resistance and virulence.

Colistin (polymyxin E) is a group of cationic antimicrobial cyclic peptides and is recognized as a last-resort defense against lethal infections with carbapenem-resistant pathogens. In addition to the plasmid-borne mobilized phosphoethanolamine (PEA) transferases, the functional expression of lipid A-modifying enzymes encoded on chromosomes has been attributed to intrinsic bacterial colistin resistance. However, the mechanisms of colistin resistance in Riemerella anatipestifer remain unknown. Herein, the GE296_RS09715 gene-encoded Lipid A PEA transferases (RaEptA) was identified in R. anatipestifer. Genetic and structural analyses revealed that the amino acid sequence of RaEptA shared 26.6%-33.1% similarities with the family of Lipid A PEA transferases (EptA) and MCR-like proteins and have defined 12 residues that contribute to the formation of phosphatidylethanolamine (PE)-recognizable cavities. Comparative analyses of colistin resistance in RA-LZ01 and RA-LZ01ΔRaEptA showed the level of colistin has fallen from 96 µg mL-1 down to 24 ~ 32 µg mL-1 . Site-directed mutagenesis assay of the PE-binding cavity and expression of the mutants reveals that K309-rRaEptA can remodel the surface of Escherichia coli and rendering it resistant to colistin, suggesting this point-mutation of P309K is necessary for EptA-mediated lipid A modification. Moreover, the virulence of RA-LZ01ΔRaEptA was attenuated compared with RA-LZ01 both in vivo and vitro. Taken together, the results represent the RaEptA involved in the colistin resistance and pathogenicity, and the P309K mutation might alter bacterial adaptation and increase the spread of colistin resistance from R. anatipestifer to other gram-negative bacteria. The findings of this study suggest another scenario for the spread of colistin resistance genes and should be considered by a wide audience.

Functional Characterization of a Novel SMR-Type Efflux Pump RanQ, Mediating Quaternary Ammonium Compound Resistance in Riemerella anatipestifer.

Riemerella anatipestifer (R. anatipestifer) is a multidrug-resistant bacterium and an important pathogen responsible for major economic losses in the duck industry. Our previous study revealed that the efflux pump is an important resistance mechanism of R. anatipestifer. Bioinformatics analysis indicated that the GE296_RS02355 gene (denoted here as RanQ), a putative small multidrug resistance (SMR)-type efflux pump, is highly conserved in R. anatipestifer strains and important for the multidrug resistance. In the present study, we characterized the GE296_RS02355 gene in R. anatipestifer strain LZ-01. First, the deletion strain RA-LZ01ΔGE296_RS02355 and complemented strain RA-LZ01cΔGE296_RS02355 were constructed. When compared with that of the wild-type (WT) strain RA-LZ01, the mutant strain ΔRanQ showed no significant influence on bacterial growth, virulence, invasion and adhesion, morphology biofilm formation ability, and glucose metabolism. In addition, the ΔRanQ mutant strain did not alter the drug resistance phenotype of the WT strain RA-LZ01 and displayed enhanced sensitivity toward structurally related quaternary ammonium compounds, such as benzalkonium chloride and methyl viologen, which show high efflux specificity and selectivity. This study may help elucidate the unprecedented biological functions of the SMR-type efflux pump in R. anatipestifer. Thus, if this determinant is horizontally transferred, it could cause the spread of quaternary ammonium compound resistance among bacterial species.

OmpA is involved in the invasion of duck brain microvascular endothelial cells by Riemerella anatipestifer.

Bacterial meningitis is a major cause of morbidity and mortality. Despite advances in antimicrobial chemotherapy, the disease remains detrimental to humans, livestock, and poultry. Riemerella anatipestifer is a gram-negative bacterium causing duckling serositis and meningitis. However, the virulence factors contributing to its binding and invasion of duck brain microvascular endothelial cells (DBMECs) and penetration of the blood-brain barrier (BBB) have never been reported. In this study, immortalized DBMECs were successfully generated and used as an in vitro-model of duck BBB. Furthermore, ompA gene deletion mutant of the pathogen and multiple complemented strains carrying the complete ompA gene and its truncated forms were constructed. Bacterial growth, invasion, and adhesion assays and animal experiments were performed. The results show that the OmpA protein of R. anatipestifer had no effect on bacterial growth and adhesion ability to DBMECs. The role of OmpA in the invasion of R. anatipestifer into DBMECs and duckling BBB was confirmed. The amino acids 230-242 of OmpA represents a key domain involved in R. anatipestifer invasion. In addition, another OmpA1164 protein constituted by the amino acids 102-488 within OmpA could function as a complete OmpA. The signal peptide sequence from amino acids 1-21 had no significant effect on OmpA functions. In conclusion, this study illustrated that OmpA is an important virulence factor mediating R. anatipestifer invasion of DBMECs and penetration of the duckling BBB.