Pathogenic Gram-negative Bacteria Research Articles

Evaluation of the Cytotoxicity Effects of Bacteriophage VbɸPA-1 on the Virulent Pseudomonas aeruginosa, A375 Human Skin Melanoma Epithelial, and HFSF PI3 Human Skin Fibroblast Cells

Background: Gram-negative bacteria are the most common infectious bacteria and are life-threatening in burn patients. Phage therapy studies can help the hospital community eliminate these bacterial pathogens. Objectives: This study aimed to evaluate the effects of the isolated bacteriophages from hospital wastewater against Gram-negative pathogenic bacteria and investigate their cytotoxicity on two human skin cell lines. Methods: The bacterial strains were isolated from burn wound infections. Bacteriophages were isolated from the hospital wastewater treatment plant in Isfahan, Iran. Transmission electron microscopy, spot tests, and restriction enzyme digestion were considered for phage identification. Finally, the lactate dehydrogenase (LDH) and tetrazolium-based MTT assays were used to realize the cytotoxicity impacts of the isolated phages. The statistical analysis of cell viability was performed using a two-way analysis of variance (ANOVA). Results: The results showed that among the isolated strains, P. aeruginosa strain NEG_RA1300 (GenBank accession number: MW845642) was sensitive to the isolated phage VbɸPA-1. The TEM results showed a possible viral taxonomy of VbɸPA-1 based on its morphology, which belonged to the Myoviridae (T4-like phages). The MTT and LDH experiments showed no cytotoxicity of VbɸPA-1 on two tested cell lines. Conclusions: Based on the results, the inhibitory effect of isolated phage on P. aeruginosa isolated from burn wounds with no toxic effect on cell lines was very significant. This phage can also be an acceptable selection against burn wound infections.

A generic cell-based biosensor converts bacterial infection signals into chemoattractants for immune cells.

Bacterial infections are a major challenge to human health. Although various potent antibiotics have emerged in the last decades, current challenges arise from an increasing number of multi-drug-resistant species. Infections associated with implants represent a particular challenge since they are usually diagnosed at an advanced state, and are difficult to treat with antibiotics due to the formation of protecting biofilms. In this study, we designed and explored a synthetic biology-inspired, cell-based bio-sensor/actor for the detection and counteraction of bacterial infections. The system is generic as it senses diverse types of infections and acts by enhancement of the endogenous immune system. The strategy is based on genetically engineered sensor/actor cells that can sense type I interferons (IFNs), which are released by immune cells at the early stages of infections. IFN signalling activates a synthetic circuit to induce reporter genes with a sensitivity of only 5 pg/ml of IFN and leads to a therapeutic protein output of 100ng/ml, resulting in theranostic cells that visualize and fight infections. Robustness and resilience were achieved by the implementation of a positive feedback loop. We show that diverse gram-positive and gram-negative implant-associated pathogenic bacteria activate the cascade in co-culture systems in a dose-dependent manner. Finally, we show that this system can be used to secrete chemoattractants facilitating the infiltration of immune cells in response to bacterial triggers. Together, the system is not only universal to bacterial infections but at the same time hypersensitive allowing the sensing of infections at initial stages.

A novel machine-learning aided platform for rapid detection of urine ESBLs and carbapenemases: URECA-LAMP.

Pathogenic gram-negative bacteria frequently carry genes encoding extended-spectrum beta-lactamases (ESBL) and/or carbapenemases. Of great concern are carbapenem resistant Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Despite the need for rapid AMR diagnostics globally, current molecular detection methods often require expensive equipment and trained personnel. Here, we present a novel machine-learning-aided platform for the rapid detection of ESBLs and carbapenemases using Loop-mediated isothermal Amplification (LAMP). The platform consists of (i) an affordable device for sample lysis, LAMP amplification, and visual fluorometric detection; (ii) a LAMP screening panel to detect the most common ESBL and carbapenemase genes; and (iii) a smartphone application for automated interpretation of results. Validation studies on clinical isolates and urine samples demonstrated percent positive and negative agreements above 95% for all targets. Accuracy, precision, and recall values of the machine learning model deployed in the smartphone application were all above 92%. Providing a simplified workflow, minimal operation training, and results in less than an hour, this study demonstrated the platform's feasibility for near-patient testing in resource-limited settings.IMPORTANCEExtended-spectrum beta-lactamases (ESBL) and carbapenemases confer resistance to third-generation cephalosporins and carbapenems in pathogenic Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Conventional antimicrobial susceptibility testing is based on phenotypic methods, and results can take several days to be obtained. Current genotypic detection methods can be rapid but require expensive equipment and trained personnel. In this study, we present a novel machine learning-aided platform for the rapid detection of ESBLs and carbapenemases using Loop-mediated isothermal Amplification (LAMP). The validation of the platform demonstrated percent positive and negative agreements above 95% for all targets. The newly developed platform provided a simplified workflow, minimal technical training, and results in less than an hour. This study demonstrated the platform's feasibility for rapid testing of ESBL and carbapenemases in bacteria and urine specimens.

Molecular insights and functional analysis of isocitrate dehydrogenase in two gram-negative pathogenic bacteria.

Klebsiella pneumoniae and Legionella pneumophila are common Gram-negative bacteria that can cause lung infections. The multidrug resistance of K. pneumoniae presents a significant challenge for treatment. This study focuses on isocitrate dehydrogenase (IDH), a key enzyme in the oxidative metabolic pathway of these two bacteria. KpIDH and LpIDH were successfully overexpressed and purified, and their biochemical characteristics were thoroughly investigated. The study revealed that KpIDH and LpIDH are homodimeric enzymes with molecular weights of approximately 70kDa. They are completely dependent on the coenzyme NADP+ and are inactive towards NAD+. KpIDH exhibits the highest catalytic activity at pH 8.0 in the presence of Mn2+ and at pH 7.8 in the presence of Mg2+. Its optimal catalytic performance is achieved with both ions at 55°C. LpIDH exhibited its highest activity at pH 7.8 in the presence of Mn2+ and Mg2+, respectively, and exhibits optimal catalytic performance at 45°C. Heat inactivation studies showed that KpIDH and LpIDH retained over 80% of their activity after being exposed to 45°C for 20min. Furthermore, we successfully altered the coenzyme specificity of KpIDH and LpIDH from NADP+ to NAD+ by replacing four key amino acid residues. This study provides a comprehensive biochemical characterization of two multidrug-resistant bacterial IDHs commonly found in hospital environments. It enhances our understanding of the characteristics of pathogenic bacteria and serves as a reference for developing new therapeutic strategies.

Facile modification of TiO2 as S-Scheme multifunctional materials for environmental protection and energy-storage applications

This paper reports a simple one-step modification of commercial TiO2 with a conducting polytrimethoxyslilypropyl aniline (PTMSPA, a polyaniline derivative having silyl networks) to have multi-functional (photocatalytic, optical, pseudocapacitive, hydrophobic, porous, antibacterial, and electromagnetic interference shielding (EMI)) properties and demonstrates associated applications. We present the S-Scheme heterojunction (SHJ) formation between TiO2 and PTMSPA through band position alignments and designate the resultant TiO2 - TiO2- based SHJ multifunctional materials as MF-TSSM. The internal electric field that is generated at the interface facilitates the transportation of the photogenerated electron transfer . The XRD pattern of MF-TSSM exhibits mainly the peaks of anatase TiO2. The TEM image of MF-TSSM indicates that the TiO2 particles are spherical in shape with sizes showing variations in diameters ranging from 60 nm to 250 nm depending on the experimental conditions of preparation and TiO2 particles are homogeneously distributed within the PTMSPA matrix. The optical energy band gap of MF-TSSM is 1.60 eV, a much lower value than PTMSPA (3.02 eV), suggesting that the composite formation between TiO2 and PTMSPA. The contact angle of MF-TSSM is significantly increased to 47.1 ° from 8.0 of TiO2, informing the increase of hydrophobic characteristics. The rate constant (k) for methylene blue photodegradation was 0.030 min-1, and 0.010 min-1 for MF-TSSM and pristine TiO2, respectively, The MF-TSSM composite exhibits a higher specific capacitance value (28.7 F/g) than TMSPA (21.1 F/g). At a frequency of 0.42 THz, MF-TSSM and PTMSPA exhibit return loss features indicating good EMI shielding performance. The MF-TSSM has been tested against two different Gram-positive and Gram-negative bacterial strains. The nanoparticles were evaluated at doses of 10, 20, 40, and 80 µg/ml. The results indicated that Klebsiella pneumoniae demonstrated a greater zone of inhibition, ranging from 9 mm at 10 µg/ml to 23 mm at 80 µg/ml, indicating increased susceptibility. Pseudomonas aeruginosa exhibited reduced inhibition zones, ranging from 10 mm at 10 µg/ml to 14 mm at 80 µg/ml, indicating increased resistance. The excellent effectiveness of MF-TSSM against various Gram-positive and Gram-negative pathogenic bacteria is explained by the interaction between reactive oxygen species and the cellular components of the bacteria as well the electrostatic interaction arising between positive charges in TMSPA and negative charges in the cell components, resulting in notable cytotoxicity and damage to the bacterial cells. The research underscores the capability of these modified TiO₂ nanoparticles in addressing bacterial infections, with efficiency differing by bacterial strain.

The outer membrane protein, OMP71, of Riemerella anatipestifer, mediates adhesion and virulence by binding to CD46 in ducks

The Riemerella anatipestifer bacterium is known to cause infectious serositis in ducklings. Moreover, its adherence to the host’s respiratory mucosa is a critical step in pathogenesis. Membrane cofactor protein (MCP; CD46) is a complement regulatory factor on the surface of eukaryotic cell membranes. Bacteria have been found to bind to this protein on host cells. Outer membrane proteins (OMPs) are necessary for adhesion, colonisation, and pathogenicity of Gram-negative bacteria; however, the mechanism by which R. anatipestifer adheres to duck cells remains unclear. In this study, pull-down assays and LC–MS/MS identified eleven OMPs interacting with duck CD46 (dCD46), with OMP71 exhibiting the strongest binding. The ability of an omp71 gene deletion strain to bind dCD46 is weaker than that of the wild-type strain, suggesting that this interaction is important. Further evidence of this interaction was obtained by synthesising OMP71 using an Escherichia coli recombinant protein expression system. Adhesion and invasion assays and protein and antibody blocking assays confirmed that OMP71 promoted the R. anatipestifer YM strain (RA-YM) adhesion to duck embryo fibroblasts (DEFs) by binding to CD46. Tests of the pathogenicity of a Δomp71 mutant strain of RA-YM on ducks compared to the wild-type parent supported the hypothesis that OMP71 was a key virulence factor of RA-YM. In summary, the finding that R. anatipestifer exploits CD46 to bind to host cells via OMP71 increases our understanding of the molecular mechanism of R. anatipestifer invasion. The finding suggests potential targets for preventing and treating diseases related to R. anatipestifer infection.

Group VA Aromatic Thiosemicarbazone Complexes: Synthesis, Characterization, Biological Activity, and Topological Studies.

The antiproliferative and antibacterial activities of thiosemicarbazones increase markedly with the presence of metal ions. One of the factors determining the activity of metal thiosemicarbazone complexes is the coordination structure. In this study, the biological effects of new antimony (III) and bismuth (III) thiosemicarbazone complexes with different binding modes and geometrical structures were demonstrated. Three new complexes, with the formulae {[SbCl3(µ2-S-Hacptsc)(η1-S-Hacptsc)], 2/3H2O,1/3CH2Cl2}, {[SbCl3(κ2-S,N-Hacpmtsc)(η1-S-Hacpmtsc)2CH2Cl2]}, and{[BiCl3(η1-S-Hbzmtsc)3]·C2H5OH}, where Hacptsc: acetophenone thiosemicarbazone, Hacpmtsc: acetophenone-N-methyl thiosemicarbazone, Hbzmtsc: benzaldehyde-N-methyl thiosemicarbazone) were elucidated by different methods and deeply analyzed in accordance with their structure by X-ray structure analysis and Atoms-In-Molecules topological analysis. This analysis provided a deeper understanding of the coordination spheres of the Sb/Bi complexes. For instance, the first reported two binding modes of the same ligand are observed in a single crystal structure of antimony (III) halide complexes. Additionally, in one of the complexes, a solid-to-solid phase transition was detected and analyzed in detail. Those complexes, very unique in terms of their geometry, have also been tested for their in vitro cytotoxic activity against human adenocarcinoma cervical cancer (HeLa) cells, whereas antimony (III) complex 1is the most active complex of this study. Further, the antibacterial activity of the complexes has been screened against two Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and two Gram-positive (Staphylococcus epidermidis and Staphylococcus aureus) pathogenic bacteria. From the results, it is found that all the complexes exhibited significant activity against the Gram-negative pathogenic bacteria.

BIOACTIVE COMPONENTS AND ANTIBACTERIAL ACTIVITY OF RAW AND BOILED EGYPTIAN PEPPER

In the current study, two cultivars of pepper (Capsicum annuum and Capsicum frutescens) at two maturity stages (green and red) were evaluated for their contents of some bioactive compounds and antibacterial activities for their ethanolic and aqueous extracts in both raw and heat-treated forms (boiling). Boiling treatment was performed under the Egyptian household conditions. Proximate analyses of the tested samples were determined, and the resulted data showed that ash and crude protein were declined after boiling treatment, while crude fat and total carbohydrate increased. Vitamin C, β-carotene and vitamin E as well as capsaicin contents were also estimated by HPLC, and the obtained data showed that all of those components were lowered by boiling treatment. Antioxidant activity of fresh and heat-treated pepper samples was carried out by using of 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS•) assay. Data showed that all pepper samples had high antioxidant activities which were increased because of boiling treatment. Finally, the antibacterial activity of all ethanolic and aqueous extracts as well as capsaicin standard was tested against both Gram-positive and Gram-negative pathogenic bacteria. Only, ethanolic extracts partially inhibited all the tested organisms except, Bacillus cereus which was completely inhibited by both ethanolic and aqueous extracts.

The aryl hydrocarbon receptor and FOS mediate cytotoxicity induced by Acinetobacter baumannii

Acinetobacter baumannii is a pathogenic and multidrug-resistant Gram-negative bacterium that causes severe nosocomial infections. To better understand the mechanism of pathogenesis, we compare the proteomes of uninfected and infected human cells, revealing that transcription factor FOS is the host protein most strongly induced by A. baumannii infection. Pharmacological inhibition of FOS reduces the cytotoxicity of A. baumannii in cell-based models, and similar results are also observed in a mouse infection model. A. baumannii outer membrane vesicles (OMVs) are shown to activate the aryl hydrocarbon receptor (AHR) of host cells by inducing the host enzyme tryptophan-2,3-dioxygenase (TDO), producing the ligand kynurenine, which binds AHR. Following ligand binding, AHR is a direct transcriptional activator of the FOS gene. We propose that A. baumannii infection impacts the host tryptophan metabolism and promotes AHR- and FOS-mediated cytotoxicity of infected cells.

Glycoengineering with neuraminic acid analogs to label lipooligosaccharides and detect native sialyltransferase activity in gram-negative bacteria.

Lipooligosaccharides (LOS) are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host-microbe interactions. Certain Gram-negative pathogenic bacteria cap their LOS with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides (LOS) at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their LOS. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the LOS of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering (MOE) and Selective Exo-Enzymatic Labeling (SEEL), and that together they provide tools to modify, label, detect and visualize sialylation of bacterial LOS.

Synthesis and characterization of a zinc-triazole coordination complex with potent antimicrobial and anticancer properties

The synthesis, characterization, and biological appraisement of a novel coordination compound [Zn(C4N4H5O)2(H2O)2]Cl have been comprehensively studied. The compound was synthesized using N-(1H-1,2,4-triazol-3-yl) acetamide and ZnCl2·6H2O, yielding a product characterized by FT-IR, elemental analysis, thermogravimetric analysis (TGA), and single crystal X-ray diffraction. FT-IR spectra confirmed the successful coordination of the ligand to the zinc ion, with significant shifts in characteristic absorption bands such as CO (1685 cm⁻¹), CN (1624 cm⁻¹), and NH (3394 cm⁻¹). Single crystal X-ray diffraction analyses revealed that the complex crystallized in the monoclinic system with space group P21/c and forms six-membered chelate rings. The unit cell parameters were a = 6.0595(3) Å, b = 14.2956(7) Å, c = 9.4762(4) Å, and β = 93.275(4)°. Thermal analysis showed that the complex undergoes stepwise decomposition with significant mass losses at various temperature intervals, including an overall mass loss of 71.09 % between 200 °C and 885 °C. Molecular docking studies indicated strong interactions between the [Zn(C4N4H5O)2(H2O)2]Cl complex and the 6QF6 cancer-related protein antigen A4, with low binding energy values (-9.32 to -8.44 kcal/mol) suggesting high binding affinity and potential biological activity. In vitro antimicrobial screening against several pathogenic microorganisms, viz. both gram-positive and gram-negative bacteria, demonstrated that the [Zn(C4N4H5O)2(H2O)2]Cl complex exhibited superior antibacterial activity, with inhibition zone diameters of 15.3 ± 0.47 mm for Bacillus subtilis and 24.17±0.56 mm for Agrobacterium tumefaciens, compared to N-(1H-1,2,4-triazol-3-yl) acetamide alone and some standard antibiotics. These findings highlight the [Zn(C4N4H5O)2(H2O)2]Cl complex as a promising candidate for developing new antimicrobial and anticancer agents, providing a foundation for further research and potential therapeutic applications.

Bioemulsifier from sponge-associated bacteria reduces staphylococcal biofilm

Biofilm formation is a major health concern and studies have been pursued to find compounds able to prevent biofilm establishment and remove pre-existing biofilms. While biosurfactants (BS) have been well-known for possessing antibiofilm activities, bioemulsifiers (BE) are still scarcely explored for this purpose. The present study aimed to evaluate the bioemulsifying properties of cell-free supernatants produced by Bacillaceae and Vibrio strains isolated from marine sponges and investigate their antiadhesive and antibiofilm activities against different pathogenic Gram-positive and Gram-negative bacteria. The BE production by the marine strains was confirmed by the emulsion test, drop-collapsing, oil-displacement, cell hydrophobicity and hemolysis assays. Notably, Bacillus cereus 64BHI1101 displayed remarkable emulsifying activity and the ultrastructure analysis of its BE extract (BE64-1) revealed the presence of structures typically observed in macromolecules composed of polysaccharides and proteins. BE64-1 showed notable antiadhesive and antibiofilm activities against Staphylococcus aureus, with a reduction of adherence of up to 100 % and a dispersion of biofilm of 80 %, without affecting its growth. BE64-1 also showed inhibition of Staphylococcus epidermidis and Escherichia coli biofilm formation and adhesion. Thus, this study provides a starting point for exploring the antiadhesive and antibiofilm activities of BE from sponge-associated bacteria, which could serve as a valuable tool for future research to combat S. aureus biofilms.

Identification and Functional Analysis of ncRNAs Regulating Intrinsic Polymyxin Resistance in Foodborne Proteus vulgaris.

Polymyxin, known as the "last line of defense" against bacterial infection, exerts a significant inhibitory effect on a wide range of Gram-negative pathogenic bacteria. The presence of strains, specifically Proteus vulgaris species, displaying intrinsic polymyxin resistance poses significant challenges to current clinical treatment. However, the underlying mechanism responsible for this intrinsic resistance remains unclear. Bacterial non-coding RNAs (ncRNAs) are abundant in genomes and have been demonstrated to have significant regulatory roles in antibiotic resistance across various bacterial species. However, it remains to be determined whether ncRNAs in Proteus vulgaris can regulate intrinsic polymyxin resistance. This study focused on investigating the foodborne Proteus vulgaris strain P3M and its intrinsic polymyxin resistance regulation mediated by ncRNAs. Through a combination of bioinformatics analysis, mutant construction, and phenotypic experimental verification, we successfully identified the ncRNAs involved and their potential target genes. These findings serve as an essential foundation for the precise identification of ncRNAs participating in the intricate regulation process of polymyxin resistance. Additionally, this study offers valuable insights into the efficient screening of bacterial ncRNAs that contribute positively to antibiotic resistance regulation.

Vicia ervilia lectin (VEA) has an antibiofilm effect on both Gram-positive and Gram-negative pathogenic bacteria

Bacterial growing resistance to antibiotics poses a critical threat to global health. This study investigates, for the first time, the antibiofilm properties of Vicia ervilia agglutinin (VEA) from six different V. ervilia accessions against pathogenic bacteria, and the yeast Candida albicans. In the absence of antimicrobial properties, purified VEA significantly inhibited biofilm formation, both in Gram-positive and Gram-negative bacteria, but not in C. albicans. With an inhibitory concentration ranging from 100 to 500 µg/ml, the VEA antibiofilm activity was more relevant against the Gram-positive bacteria Streptococcus aureus and Staphylococcus epidermidis, whose biofilm was reduced up to 50% by VEA purified from accessions #5 and #36. VEA antibiofilm variability between accessions was observed, likely due to co-purified small molecules rather than differences in VEA protein sequences. In conclusion, VEA seed extracts from the accessions with the highest antibiofilm activity could represent a valid approach for the development of an effective antibiofilm agent.

Plant-induced bacterial gene silencing: a novel control method for bacterial wilt disease.

Ralstonia pseudosolanacearum, a notorious phytopathogen, is responsible for causing bacterial wilt, leading to significant economic losses globally in many crops within the Solanaceae family. Despite various cultural and chemical control strategies, managing bacterial wilt remains a substantial challenge. This study demonstrates, for the first time, the effective use of plant-induced bacterial gene silencing against R. pseudosolanacearum, facilitated by Tobacco rattle virus-mediated gene silencing, to control bacterial wilt symptoms in Nicotiana benthamiana. The methodology described in this study could be utilized to identify novel phytobacterial virulence factors through both forward and reverse genetic approaches. To validate plant-induced gene silencing, small RNA fractions extracted from plant exudates were employed to silence bacterial gene expression, as indicated by the reduction in the expression of GFP and virulence genes in R. pseudosolanacearum. Furthermore, treatment of human and plant pathogenic Gram-negative and Gram-positive bacteria with plant-generated small RNAs resulted in the silencing of target genes within 48 hours. Taken together, the results suggest that this technology could be applied under field conditions, offering precise, gene-based control of target bacterial pathogens while preserving the indigenous microbiota.

The effect of selenium on the intestinal health of juvenile grass carp based on the ERS-autophagy pathway

Selenium (Se), a trace element, is vital for the maintenance of cellular redox balance, thyroid hormone metabolism, inflammation, and immunity. Aeromonas hydrophila (A. hydrophila) is a common Gram-negative conditional pathogenic bacterium in fish culture, posing a serious threat to intensive aquaculture. Our study investigated the influence of dietary Se on the intestinal immune function of grass carp (Ctenopharyngodon idella) and the related regulatory mechanisms. The 2160 healthy juvenile grass carp (9.76 ± 0.005 g) were randomly assigned to 6 test groups of 6 replicates each, and fed graded selenomethionine (0.05, 0.20, 0.40, 0.61, 0.77, 0.98 mg Se/kg diet) for 70 days and then injected with A. hydrophila for a 6-day attack test. The results indicated that appropriate Se levels (0.40 mg/kg diet) alleviated intestinal damage caused by A. hydrophila and increased intestinal immune substances C3 and C4 levels as well as the activity of acid phosphatase (ACP) and lysozyme (LZ) (P > 0.05). Appropriate levels of Se (0.40 mg/kg-0.61 mg/kg diet) decreased intestinal pro-inflammatory cytokines (IFN-γ2, IL-6, IL-12p35, IL-17 A F and IL-17D) mRNA levels (P > 0.05) and increased intestinal anti-inflammatory factors (TGF-β1, IL-4/13A, IL-4/13B, IL-10 and IL-22) mRNA levels (P > 0.05) in juvenile grass carp. Further studies revealed that Se (0.40 mg/kg-0.61 mg/kg diet) inhibited intestinal endoplasmic reticulum stress (ERS)-related signaling pathway. Furthermore, we found that appropriate levels of Se (0.40 mg/kg-0.61 mg/kg diet) inhibited intestinal autophagy in juvenile grass carp, which may be related to ULK1, Beclin 1, ATG5, ATG12, LC3, and P62. In conclusion, appropriate levels of Se can alleviate intestinal inflammation and inhibit ERS and autophagy in juvenile grass carp. A quadratic regression analysis of intestinal ACP and LZ also indicated that the Se requirements of juvenile grass carp were 0.59 and 0.51 mg/kg, respectively.

Identifikasi Bakteri Patogen Gram Negatif pada Alat Praktikum Mikrobiologi di Laboratorium Central Stikes Maharani Malang

Stacks of laboratory equipment and lack of routine sterilization can contribute to the growth and contamination of pathogenic bacteria. This research aimed to determine the types of gram-negative pathogenic bacteria present in the microbiology practicum equipment at the Central STIKes Maharani Malang Laboratory. This type of research is descriptive research with an analytical research design. The sampling technique uses purposive sampling. This research involves taking swab samples on the surface of practical microbiology equipment, isolating bacterial colonies using MCA (Mac Conkey Agar) media, making pure cultures on MCA slanted agar media, macroscopic observations, and biochemical tests. Bacteria were grown on MCA media for 2x24 hours. The results of research using gram staining showed gram-negative bacteria, rod-shaped, spread, and red. Biochemical tests showed glucose (+g), lactose (+), mannitol (+), maltose (+), sucrose (+), H2S (-), indole (-), Mr (Methyl Red) (-), Vp ( Voges Praskaeur) (+), citrate (+), motility (-), the bacterial species Klebsiella pneumoniae was found. The second result shows glucose (+g), lactose (+), mannitol (+), maltose (+), sucrose (+), H2S (-), indole (+), Mr (Methyl Red) (+), Vp ( Voges Praskaeur) (-), citrate (-), motility (-), found Escherichia coli bacterial species. This study concluded that it was found that gram-negative bacteria were rod-shaped, spread, and red. Meanwhile, from biochemical tests using glucose, lactose, mannitol, maltose, sucrose, ındol, MR, VP, and citrate, Klebsiella pneumoniae and Escherichia coli bacteria were found.

A phosphodiesterase CpdB in Yersinia pseudotuberculosis degrades CDNs to inhibit innate immune response

Yersinia pseudotuberculosis (Yptb) is a pathogenic gram-negative bacterium that can colonize the intestines of different animals. Its infection leads to the activation of the host’s innate immunity. Both host and bacterial-derived cyclic dinucleotides (CDNs) could activate the innate immune response of host cells. In bacteria, CDNs like c-di-AMP, c-di-GMP, or 3′3'-cGAMP can be hydrolyzed by different hydrolases. Recent studies showed that the degradation of those second messengers helps the pathogen evade immune detection. In this study, we identified a hydrolase, YPK_3776, namely CpdB in Yptb. CpdB is predicted to bind bacterial-derived c-di-AMP, c-di-GMP, 3′3'-cGAMP and host-derived 2′3'-cGAMP. Surprisingly, by using high-performance liquid chromatography (HPLC), we found that CpdB could only degrade bacterial-derived CDNs but not host-derived 2′3'-cGAMP. In addition, CpdB has 2′3'-cNMP activity. Consistently, the Yptb mutant lacking the cpdB gene exhibited a higher level of intracellular c-di-GMP. Furthermore, the ∆cpdB mutant elicited stronger innate immune responses during Yptb infection in macrophages, suggesting CpdB enables Yptb to evade host immune surveillance. Furthermore, CpdB inhibited the Yptb-induced innate immune response in a STING-dependent manner. Finally, we showed the ∆cpdB infection in mice model exhibited in lower bacterial burden, as compared to wild-type strain infection, indicating CpdB is important for bacterial survival in the host. Together, we identified a cyclic dinucleotide hydrolase CpdB in Yptb that could degrade bacterial-derived CDNs which help the pathogen to evade immune detection via the STING pathway.

Quantum dots as a fluorescent labeling tool for live-cell imaging of Leptospira.

Leptospirosis is a global public health problem caused by Gram-negative pathogenic bacteria belonging to the genus Leptospira. The disease is transmitted through the urine of infected animals, which contaminates water and soil, leading to the infection of other animals and humans. Currently, several approaches exist to detect these bacteria; however, a new sensitive method for the live-cell imaging of Leptospira is required. In this study, we report the green synthesis of cadmium telluride quantum dots (CdTe QDs) which are unique fluorescent nanocrystals with a high fluorescence quantum yield capable of modifying cell surfaces and are biocompatible with cells. The fabrication of QDs with concanavalin A (ConA), a carbohydrate-binding lectin and known biological probe for Gram-negative bacteria, produced ConA-QDs which can effectively bind on Leptospira and exhibit strong fluorescence under simple fluorescence microscopy, allowing the live-cell imaging of the bacteria. Overall, we performed the simple synthesis of ConA-QDs and demonstrated their potential use as versatile fluorescent probes for the live-cell imaging of Leptospira. This technique could be further applied to track leptospiral cells and study the infection mechanism, contributing to a more thorough understanding of leptospirosis and how to control it in the future.

Detection of Molecular Vibrations of Shigella Pathogenic Gram-negative Bacterium with Surface Enhanced Raman Spectroscopy (SERS) Biosensors and Investigation of its Antibacterial Activity with Silver Nanoparticles Prepared by the Tollens Method in a Laboratory Environment

Detection of Molecular Vibrations of Shigella Pathogenic Gram-negative Bacterium with Surface Enhanced Raman Spectroscopy (SERS) Biosensors and Investigation of its Antibacterial Activity with Silver Nanoparticles Prepared by the Tollens Method in a Laboratory Environment